Aryl imidazoles and their use as anti-cancer agents

ABSTRACT

Therapeutically effective 2,4,5-trisubstituted imidazole compounds are provided. Also provided are methods of preparing the compounds and pharmaceutical compositions comprising the compounds alone or in combination with other agents. The present invention further provides for the use of the compounds as anti-cancer agents; wherein: R1 is aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl or amino; R2 and R3 are independently aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 and R3 when taken together along with the carbon atoms they are attached to, form aryl or substituted aryl, and R4 is hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, aryl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or —S(O)o.2R wherein R is alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, or substituted heteroaryl.

FIELD OF INVENTION

This invention pertains to the field of anti-cancer compounds and, inparticular, to the use of therapeutically active 2,4,5-trisubstitutedimidazole compounds in the treatment of cancer.

BACKGROUND OF THE INVENTION

A cancer is a malignant tumour of potentially unlimited growth. It isprimarily the pathogenic replication (a loss of normal regulatorycontrol) of various given types of cells found in the human body. Byselect mutation resulting from a primary lesion, the DNA of a cancercell evolves and converts the cell into an autonomous system.Conventional cancer treatments have focused mainly on killing cancerouscells. Chemotherapeutic agents currently used foranti-cancer/anti-tumour therapy are selected for their toxicity towardsrapidly proliferating cells. Most of them cause undesirable systemiceffects such as cardiac or renal toxicity, marrow aplasia, alopecia,nausea and vomiting. During the last few years, many researchers havetried to eliminate these side effects by developing drugs havingsuitable physico-chemical properties allowing an increase of theavailability of the drug to the tumour site. New molecules extractedfrom natural sources, synthetically or semi-synthetically produced,enzymes, radioisotopes, DNA toxins, various macromolecules, andantibodies against fibrin or against tumour-specific surface antigensare bound to drugs in an attempt to increase selectivity of thechemotherapeutic agents.

The effectiveness of most anticancer agents is greatly reduced becauseof their high toxicity and the nature of the illness. It is believedthat the problem of high toxicity of the anticancer agents can becircumvented by chemical modifications of those structures in such a waythat they act more specifically on tumour cells without increasingsystemic toxicity. The research in this field is therefore mainlydirected to the synthesis of anticancer agents which would possess highantineoplastic activity, low systemic toxicity and low mutagenicity onnormal cells.

Heterocyclic compounds, especially heterocyclic azole derivatives, havebeen shown to have a wide spectrum of biological activities. One classof compounds with interesting biological activities is the imidazoles(derivatives containing a five-membered heterocyclic azole). A varietyof biological activities have been reported for imidazole derivativeswith different substitution patterns (Lee et al. Nature 1994327:739-745; Abdel-Meguid el al. Biochemistry, 1994, 33:11671; Heerdinget al. Bioorg. Med. Chem. Lett. 2001, 11:2061-2065; Bu et al.Tetrahedron Lett. 1996, 37:7331-7334; Lewis J R. Nat. Prod. Rep. 1999,16:389-418; Lewis J R. Nat. Prod. Rep. 1998, 15:417-437 and 371-395).

Biological activities have also been reported for aryl-imidazolederivatives, for example, these compounds can act as modulators ofmulti-drug resistance in cancer cells (Zhang et al. Bioorg. Med. Chem.Lett. 2000, 10:2603-2605), inhibitors of p38 MAP kinase (Adams et al.Bioorg. Med. Chem. Lett. 2001, 11:867-2870, McLay et. al. Bioorg. Med.Chem. 2001, 9:537-554) and of cytokines (U.S. Pat. Nos. 5,656,644;5,686,455; 5,916,891; 5,945,418; and 6,268,370), and inhibitors ofbacterial growth (Antolini et al. Bioorg. Med. Chem. Lett. 1999,9:1023-1028).

A few reports have indicated that triaryl-imidazole compounds can act asinhibitors of p38 MAP kinase (for example, see LoGrasso et al.Biochemistry, 1997, 36:10422-10427) and as modulators of multi-drugresistance in cancer cells (Sarshar et al. Bioorg. Med. Chem. Lett.2000, 10:2599-2601), however, the majority of the literature indicatesthat these compounds have found use mainly as colour producing reagents(U.S. Pat. Nos. 4,089,747; 5,024,935; 5,047,318; 5,496,702; 5,514,550;and 5,693,589) and as photopolymerization initiators (U.S. Pat. Nos.6,117,609 and 6,060,216), generally in dimeric form.

The potential anti-cancer activity of a number of compounds has beeninvestigated by the National Cancer Institute (NCI), which hasundertaken a large scale screening of several thousand compounds to tryto identify those that have potential therapeutic application in thetreatment of cancer (NCI Yeast Anticancer Drug Screen). The screen isbased on the ability of candidate compounds to inhibit the growth ofSaccharmyces cerevisiae strains that have mutations in genes related tocell cycle control and DNA repair damage. Compounds are initiallyscreened against a panel of six yeast strains at a single concentration(Stage0). Compounds with activity in Stage0 are re-screened against thesame panel at two concentrations (Stage1). Selected compounds withactivity in Stage1 that also show selectivity are re-screened against apanel of 13 yeast strains at five concentrations (Stage2). Many of theresults from the screening have been made available on the NCI/DTPwebsite. The approach adopted in this screen is dependent on a candidatecompound exerting its activity on certain cellular pathways (i.e. cellcycle control or DNA repair damage). The results generated by this typeof screen, therefore, represent a very preliminary stage of screeningfor potential anti-cancer drugs and do not necessarily correlate withthe ability of a compound to inhibit the growth of cancer cells in vitroor in vivo.

The NCI also provides an in vivo screening program to try to identifypotential anti-cancer drugs (NCI In Vivo Anticancer Drug Screen). Manyof the results from this screening program are also available from theNCI/DTP website.

Amongst those compounds tested in one or both of the NCI screens aresome aryl imidazole compounds (NCI #322334, 338970, 144033). None ofthese three compounds showed any activity in the In Vivo Anticancer DrugScreen, even though one of these compounds (NCI #338970) had beenreported as active in Stage0 testing in the Yeast Anticancer DrugScreen. The fact that this compound was active in the yeast screen yetshowed no activity in the in vivo assay confirms that a positive resultin the yeast screen is not necessarily predictive of the utility of acompound as in anti-cancer therapeutic.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a class of compoundswhich are 2,4,5-trisubstituted imidazole derivatives that haveanti-cancer activity. In accordance with an aspect of the presentinvention there is provided a use of a compound having structuralformula (I), or a salt thereof, as an anti-cancer agent:

wherein:

R1 is aryl, substituted aryl, heterocycle, substituted heterocycle,heteroaryl, substituted heteroaryl or amino;

R2 and R3 are independently aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 andR3 when taken together along with the carbon atoms they are attached to,form aryl or substituted aryl, heterocycle, substituted heterocycle,heteroaryl, or substituted heteroaryl and

R4 is hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, substituted alkylthiol, acyl, aryloxy, amino, amido,carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substitutedheterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl,alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano, —S(O)₀₋₂R whereinR is alkyl, substituted alkyl, aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl.

In accordance with another aspect of the present invention, there isprovided a use of a compound having structural formula (I), or a saltthereof, in the preparation of an anti-cancer composition.

In accordance with another aspect of the present invention, there isprovided a compound having the structural formula:

or a salt thereof, wherein:

R2 and R3 are independently aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 andR3 when taken together along with the carbon atoms they are attachedform aryl or substituted aryl, heterocycle, heteroaryl, substitutedheterocycle, or substituted heteroaryl;

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, —CH₂-aryl, —CH₂-heteroaryl.

In accordance with another aspect of the present invention, there isprovided a compound having the structural formula:

or a salt thereof wherein:

Ph1 and Ph2 are independently selected from phenyl and substitutedphenyl;

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, low alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl; R10 is H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, —CH₂-aryl, —CH₂-heteroaryl.

In accordance with another aspect of the present invention, there isprovided a compound having the structural formula:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl; R10 is H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, —CH₂-aryl, —CH₂-heteroaryl;

x is CR11 or N;

y is CR12 or N;

z is CR13 or N;

r is CR14 or N;

x′ is CR15 or N;

y′ is CR16 or N;

z′ is CR17 or N;

r′ is CR18 or N;

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio,acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In accordance with another aspect of the present invention, there isprovided a compound having the structural formula:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, —CH₂-aryl, —CH₂-heteroaryl;

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio,acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In accordance with another embodiment of the present invention, there isprovided a use of a therapeutically effective amount of a compound offormula I:

wherein:

-   -   R1 is aryl, substituted aryl, heterocycle, substituted        heterocycle, heteroaryl, substituted heteroaryl or amino;    -   R2 and R3 are independently aryl, substituted aryl, heterocycle,        heteroaryl, substituted heterocycle, or substituted heteroaryl        or R2 and R3 when taken together along with the carbon atoms        they are attached to, form aryl or substituted aryl, and    -   R4 is hydrogen, halogen, hydroxyl, thiol, lower alkyl,        substituted lower alkyl, lower alkenyl, substituted lower        alkenyl, lower alkynyl, substituted lower alkynyl, alkylalkenyl,        alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido,        carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,        substituted heterocycle, heteroalkyl, cycloalkyl, substituted        cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano        or —S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl,        substituted aryl, heterocycle, heteroaryl, substituted        heterocycle, or substituted heteroaryl,        to inhibit neoplastic cell growth or proliferation in a mammal.

In accordance with another embodiment of the present invention, there isprovided a use of a therapeutically effective amount of a compound offormula I:

wherein:

-   -   R1 is aryl, substituted aryl, heterocycle, substituted        heterocycle, heteroaryl, substituted heteroaryl or amino;    -   R2 and R3 are independently aryl, substituted aryl, heterocycle,        heteroaryl, substituted heterocycle, or substituted heteroaryl        or R2 and R3 when taken together along with the carbon atoms        they are attached to, form aryl or substituted aryl, and    -   R4 is hydrogen, halogen, hydroxyl, thiol, lower alkyl,        substituted lower alkyl, lower alkenyl, substituted lower        alkenyl, lower alkynyl, substituted lower alkynyl, alkylalkenyl,        alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido,        carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,        substituted heterocycle, heteroalkyl, cycloalkyl, substituted        cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano        or —S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl,        substituted aryl, heterocycle, heteroaryl, substituted        heterocycle, or substituted heteroaryl,        in the treatment of cancer in a mammal in need thereof.

In accordance with another embodiment of the present invention, there isprovided a compound selected from the compounds of structural formulae:

wherein:

-   -   R2 and R3 are independently aryl, substituted aryl, heterocycle,        heteroaryl, substituted heterocycle, or substituted heteroaryl        or R2 and R3 when taken together along with the carbon atoms        they are attached to, form a aryl, substituted aryl,        heterocycle, heteroaryl, substituted heterocycle, or substituted        heteroaryl;    -   R4, R5, R6, R7, R8 and R9 are independently selected from        hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted        lower alkyl, lower alkenyl, substituted lower alkenyl, lower        alkynyl, substituted lower alkynyl, alkylalkenyl, alkyl alkynyl,        alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,        substituted aryl, heterocycle, heteroaryl, substituted        heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl,        alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or        —S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl,        substituted aryl, heterocycle, heteroaryl, substituted        heterocycle, or substituted heteroaryl.

In accordance with another embodiment of the present invention, there isprovided a compound selected from the compounds of structural formulae:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

x is CR11 or N;

y is CR12 or N;

z is CR13 or N;

r is CR14 or N;

x′ is CR15 or N;

y′ is CR16 or N;

z′ is CR17 or N;

r′ is CR18 or N;

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In accordance with another embodiment of the present invention, there isprovided a use of a compound of formula (I) in the manufacture of amedicament for the inhibition of neoplastic cell growth orproliferation.

In accordance with another embodiment of the present invention, there isprovided a use of a compound of formula (I) in the manufacture of amedicament for the treatment of cancer.

In accordance with another aspect of the present invention, there isprovided an anti-cancer composition comprising an effective amount of acompound having structural formula (I), or a salt thereof, and acarrier, diluent or excipient.

In accordance with another aspect of the present invention there isprovided a method of inhibiting neoplastic cell growth or proliferationin a mammal comprising administering to said mammal a therapeuticallyeffective amount of a compound selected from the compounds of generalformula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X),(XI), (XII) and (XII), or a salt thereof.

In accordance with another aspect of the present invention there isprovided a method of treating cancer in a mammal comprisingadministering to said mammal a therapeutically effective amount of acompound selected from the compounds of general formula (I), (II),(III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII) and(XIII), or a salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the effects of a compound 92 on the proliferation ofvarious cancer cell lines in vitro.

FIG. 2 depicts the effects of a compound 28 on the proliferation ofvarious cancer cell lines in vitro.

FIG. 3 depicts the effects of a compound 50 on the proliferation ofvarious cancer cell lines in vitro.

FIG. 4 depicts the effects of a compound 42 on the proliferation ofvarious cancer cell lines in vitro.

FIG. 5A-C depicts the effects of various concentrations of a compound 45on the proliferation of cancer cell lines in vitro at different timeintervals.

FIG. 6A-C depicts the effects of various concentrations of a compound 45on the proliferation of cancer cell lines in vitro at different timeintervals.

FIG. 7 depicts the effects of compounds 83 and 99 on the proliferationof LS 513 colon carcinoma cells in vitro.

FIG. 8 depicts the effects of compounds of Formula I on theproliferation of HT-29 colon adenocarcinoma cells in vitro.

FIG. 9A-C present the cancer cell lines used to in the NCI screen usedto determine the ability of compounds of Formula I to inhibit cancercell proliferation in vitro.

FIG. 10A depicts the average and mean GI₅₀ values for various compoundsof Formula I for a number of cancer cell lines; B depicts the averageGI₅₀ values for compound 45 by cancer cell type and C depicts theaverage total growth inhibition (TGI) for compound 45 by cancer celltype.

FIG. 11 depicts the inhibition of H460 NSCLC cell proliferation in vitroby compounds of Formula I.

FIG. 12 depicts the inhibition of HT-29 colon carcinoma cellproliferation in vitro by compounds of Formula I.

FIG. 13 depicts the inhibition of HT-29 colon carcinoma cellproliferation in vitro by compounds of Formula I.

FIG. 14 depicts the effects of compounds of Formula I on the growth ofHT-29 colon adenocarcinoma cells in vivo in CD-1 nude mice.

FIG. 15 depicts the effects of compounds of Formula I on the averageweight of tumours in CD-1 nude mice (average weight per group of mice).

FIG. 16 depicts the effects of compounds of Formula I on the weight oftumours in CD-1 nude mice (individual tumour weights).

FIG. 17 depicts the effects of compounds of Formula I on the growth ofHT-29 colon adenocarcinoma cells in vivo in CD-1 nude mice.

FIG. 18 depicts the effect of compound 45 on the growth of HepG2hepatocarcinoma cells in vivo in CD-1 nude mice in terms of A tumoursize, and B tumour weight.

FIG. 19 depicts the effects of compounds 45, 33 and 90 on the activityof various human kinases.

FIG. 20 depicts the subcellular location of compound 45 in HT-29 colonadenocarcinoma cells (A, B); of doxorubicin in HT-29 colonadenocarcinoma cells (C); of compound 45 in A498 renal cancer cells (D),and of compound 45 in C8161 melanoma cells (E).

FIG. 21 depicts the formation of vacuoles in HT-29 colon adenocarcinomacells treated with compound 45 or doxorubicin.

FIG. 22 depicts the effects of compound 45 on the cell cycle in HT-29colon adenocarcinoma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a class of 2,4,5-trisubstituted imidazolecompounds and for their use as anti-cancer agents. The present inventionfurther provides for methods of inhibiting neoplastic cell growth and/orproliferation in an animal by administering to the animal an effectiveamount of a compound of Formula I, either alone or in combination withone or more standard chemotherapeutics.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

The terms are defined as follows:

The term “halogen” refers to fluorine, bromine, chlorine, and iodineatoms.

The term “hydroxyl” refers to the group —OH.

The term “thiol” or “mercapto” refers to the group —SH, and —S(O)₀₋₂.

The term “lower alkyl” refers to a straight chain or branched alkylgroup of one to ten carbon atoms or a cyclic alkyl group of three to tencarbon atoms. This term is further exemplified by such groups as methyl,ethyl, n-propyl, i-propyl, n-butyl, t-butyl, 1-butyl (or2-methylpropyl), cyclopropylmethyl, i-amyl, n-amyl, hexyl and the like.

The term “substituted lower alkyl” refers to lower alkyl as justdescribed including one or more groups such as hydroxyl, thiol,alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl,substituted cycloalkyl, heterocycle, cycloheteroalkyl, substitutedcycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy,hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl,alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, nitro, cyano.These groups may be attached to any carbon atom of the lower alkylmoiety.

The term “lower alkenyl” refers to a straight chain or branchedhydrocarbon of two to ten carbon atoms or a cyclic hydrocarbon of threeto ten carbon atoms, having at least one carbon to carbon double bond.

The term “substituted lower alkenyl” refers to lower alkenyl as justdescribed including one or more groups such as hydroxyl, thiol,alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl,substituted cycloalkyl, heterocycle, cycloheteroalkyl, substitutedcycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy,hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl,alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkylcycloheteroalkyl, nitro, cyano. These groups maybe attached to anycarbon atom to produce a stable compound.

The term “lower alkynyl” refers to a straight chain or branchedhydrocarbon of two to ten carbon atoms having at least one carbon tocarbon triple bond.

The term “substituted lower alkynyl” refers to lower alkynyl as justdescribed including one or more groups such as hydroxyl, thiol,alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl,substituted cycloalkyl, heterocycle, cycloheteroalkyl, substitutedcycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy,hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl,alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkylcycloheteroalkyl, nitro, cyano. These groups may be attached to anycarbon atom to produce a stable compound.

The term “alkoxy” refers to the group —OR, where R is lower alkyl,substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl,substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, or substitutedcycloheteroalkyl as defined below.

The term “alkylthio” denotes the group —SR, —S(O)_(n=1-2)—R, where R islower alkyl, substituted lower alkyl, aryl, substituted aryl, aralkyl orsubstituted aralkyl as defined below.

The term “acyl” refers to groups —C(O)R, where R is hydrogen, loweralkyl, substituted lower alkyl, aryl, substituted aryl, cycloalkyl orsubstituted cycloalkyl.

The term “aryloxy” refers to groups —OAr, where Ar is an aryl,substituted aryl, heteroaryl, or substituted heteroaryl group as definedbelow.

The term “amino” refers to the group NRR′, where R and R′ mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, heteroaryl, cycloalkyl, or substituted heteroaryl asdefined below, acyl, D or L aminoacid or a protected form thereof.

The term “amido” refers to the group —C(O)NRR′, where R and R′ mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, hetaryl, substituted hetaryl as defined below.

The term “carboxyl” refers to the group —C(O)OR, where R mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, hetaryl, substituted hetaryl and the like as defined.

The terms “aryl” or “Ar” refer to an aromatic carbocyclic group havingat least one aromatic ring (e.g., phenyl or biphenyl) or multiplecondensed rings in which at least one ring is aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, phenanthryl, 9-fluorenyl,dibenzocycloheptatrienyl etc.).

The term “substituted aryl” refers to aryl optionally substituted withone or more functional groups, e.g., halogen, hydroxyl, thiol, loweralkyl, substituted lower alkyl, trifluoromethyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, substitutedheterocycle, heteroaryl, substituted heteroaryl, heteroalkyl,substituted heteroalkyl, cycloalkyl, substituted cycloalkyl,alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido, cyano or—N═CRR′, wherein R and R′ are independently selected from H, alkyl,substituted alkyl, aryl, substituted aryl, heterocycle, substitutedheterocycle, heteroaryl or substituted heteroaryl.

The term “heterocycle” refers to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring (e.g., morpholino, pyridyl orfuryl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl,quinolinyl, indolizinyl, indanyl or benzo[b]thienyl) and having at leastone hetero atom, such as N, O or S, within the ring.

The term “substituted heterocycle” refers to heterocycle optionallysubstituted with, halogen, hydroxyl, thiol, lower alkyl, substitutedlower alkyl, trifluoromethyl, lower alkenyl, substituted lower alkenyl,lower alkynyl, substituted lower alkynyl, alkylalkenyl, alkyl alkynyl,alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl,substituted heteroaryl, heteroalkyl, substituted heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.

The terms “heteroaryl” or “hetaryl” refer to a heterocycle in which atleast one heterocyclic ring is aromatic.

The term “substituted heteroaryl” refers to a heterocycle optionallymono or poly substituted with one or more functional groups, e.g.,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl,trifluoromethyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, substituted heterocycle, heteroaryl, substitutedheteroaryl, heteroalkyl, substituted heteroalkyl, cycloalkyl,substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro,sulfamido, cyano or —N═CRR′ wherein R and R′ are independently selectedfrom H, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle,substituted heterocycle, heteroaryl or substituted heteroaryl and thelike.

The term “aralkyl” refers to the group -R—Ar where Ar is an aryl groupand R is lower alkyl or substituted lower alkyl group. Aryl groups canoptionally be unsubstituted or substituted with, e.g., halogen, loweralkyl, alkoxy, alkyl thio, trifluoromethyl, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term “heteroalkyl” refers to the group -R-Het where Het is aheterocycle group and R is a lower alkyl group. Heteroalkyl groups canoptionally be unsubstituted or substituted with e.g., halogen, loweralkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido,carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substitutedhetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term “heteroarylalkyl” refers to the group -R-HetAr where HetAr isan heteroaryl group and R lower alkyl or substituted lower alkyl.Heteroarylalkyl groups can optionally be unsubstituted or substitutedwith, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy,alkylthio, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term “cycloalkyl” refers to a cyclic or polycyclic alkyl groupcontaining 3 to 15 carbon. For polycyclic groups, these may be multiplecondensed rings in which one of the distal rings may be aromatic (e.g.tetrahydronaphthalene, etc.).

The term “substituted cycloalkyl” refers to a cycloalkyl groupcomprising one or more substituents with, e.g. halogen, hydroxyl, thiol,lower alkyl, substituted lower alkyl, trifluoromethyl, lower alkenyl,substituted lower alkenyl, lower-alkynyl, substituted lower alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido orcyano and the like.

The term “cycloheteroalkyl” refers to a cycloalkyl group wherein one ormore of the ring carbon atoms is replaced with a heteroatom (e.g., N, O,S or P).

The term “substituted cycloheteroalkyl” refers to a cycloheteroalkylgroup as herein defined which contains one or more substituents, such ashalogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl,amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl,substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and thelike.

The term “alkyl cycloalkyl” refers to the group -R-cycloalkyl wherecycloalkyl is a cycloalkyl group and R is a lower alkyl or substitutedlower alkyl. Cycloalkyl groups can optionally be unsubstituted orsubstituted with e.g. halogen, lower alkyl, lower alkoxy, loweralkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl,aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano,alkylthio, thiol, sulfamido and the like.

The terms “therapy” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of alleviating thesymptoms associated with, preventing the development of, or altering thepathology of a disease, disorder or condition. Thus, the terms therapyand treatment are used in the broadest sense, and include the prevention(prophylaxis), moderation, reduction, and curing of a disease, disorderor condition at various stages. Those in need of therapy/treatmentinclude those already having the disease, disorder or condition as wellas those prone to, or at risk of developing, the disease, disorder orcondition and those in whom the disease, disorder or condition is to beprevented.

The term “subject” or “patient,” as used herein, refers to an animal inneed of treatment.

The term “animal,” as used herein, refers to both human and non-humananimals, including, but not limited to, mammals, birds and fish.

Administration of the compounds of the invention “in combination with”one or more further therapeutic agents, is intended to includesimultaneous (concurrent) administration and consecutive administration.Consecutive administration is intended to encompass various orders ofadministration of the therapeutic agent(s) and the compound(s) of theinvention to the subject.

As used herein, the term “about” refers to a +/−10% variation from thenominal value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

I. 2,4,5-Trisubstituted Imidazole Compounds

The present invention provides compounds of the general formula (I):

or a salt thereof; wherein:

R1 is aryl, substituted aryl, heterocycle, substituted heterocycle,heteroaryl, substituted heteroaryl or amino;

R2 and R3 are independently aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 andR3 when taken together along with the carbon atoms they are attached to,form aryl or substituted aryl, heterocycle, substituted heterocycle,heteroaryl, or substituted heteroaryl;

R4 is hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, cyano or —S(O)₀₋₂R wherein R is alkyl,substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, or substituted heteroaryl.

In another embodiment of the present invention, the compound of formula(I) is other than Nortopsentin A, Nortopsentin B, Nortopsentin C andNortopsentin D.

In another embodiment of the present invention, the compound of FormulaI includes the compound of the structural formula:

or a salt thereof, wherein:

R2 and R3 are independently aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 andR3 when taken together along with the carbon atoms they are attachedform aryl or substituted aryl, heterocycle, heteroaryl, substitutedheterocycle, or substituted heteroaryl;

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, —CH₂-aryl, —CH₂-heteroaryl.

In another embodiment of the invention, the compound of Formula II isother than Nortopsentin A, Nortopsentin B, Nortopsentin C andNortopsentin D.

In another embodiment of the present invention, the compound of FormulaII includes the compound of the structural formula III:

or a salt-thereof, wherein:

Ph1 and Ph2 are independently selected from phenyl and substitutedphenyl;

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alknyyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl.

In another embodiment of the invention, the compound of Formula III isselected from:

or a salt thereof wherein:

R5, R6, R9, R11, R12 and R13 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl, alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl.

In another embodiment of the present invention, the compound of FormulaI includes the compound of the structural formula:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

x is CR11 or N;

y is CR12 or N;

z is CR13 or N;

r is CR14 or N;

x′ is CR15 or N;

y′ is CR16 or N;

z′ is CR17 or N;

r′ is CR18 or N;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl,

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In another embodiment of the present invention, the compound of FormulaI includes the compound of the structural formula:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

R10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl;

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In another embodiment of the present invention, the compound of FormulaI includes the compound of the structural formula:

wherein:

R2 and R3 are independently aryl, substituted aryl, heterocycle,heteroaryl, substituted heterocycle, or substituted heteroaryl or R2 andR3 when taken together along with the carbon atoms they are attached to,form a aryl, substituted aryl, heterocycle, heteroaryl, substitutedheterocycle, or substituted heteroaryl;

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl.

In another embodiment of the present invention, the compound of FormulaI includes the compound of the structural formula:

or a salt thereof, wherein:

R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower alkynyl, substituted loweralkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy,amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl;

x is CR11 or N;

y is CR12 or N;

z is CR13 or N;

r is CR14 or N;

x′ is CR15 or N;

y′ is CR16 or N;

z′ is CR17 or N;

r′ is CR18 or N;

R11, R12, R13, R14, R15, R16, R17 and R18 are independently selectedfrom hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In another embodiment, in the compounds of formula (XI) at least one ofR11 to R18 is other than H.

In another embodiment, in the compounds of formula (XI) at least one ofx, y, z, r, x′, y′, z′ or r′ is nitrogen.

In another embodiment the compound of formula (XI) is other than:

2-phenyl-1H-phenanthro[9,10-d]imidazole;

2-(2-methylphenyl)-1H-phenanthro[9,10-d]imidazole;

2-(3-iodophenyl)-1H-phenanthro[9,10-d]imidazole;

2-(4-dimethylaminophenyl)-1H-phenanthro[9,10-d]imidazole;

2-(4-nitrophenyl)-1H-phenanthro[9,10-d]imidazole;

1,2-diphenyl-1H-phenanthro[9,10-d]imidazole.

In another embodiment of the invention, the compound of Formula I isselected from:

wherein:

R4, R5, R6, R7, R8, R9, R11, R12 and R13 are independently selected fromhydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, cyano or —S(O)₀₋₂R wherein R is alkyl,substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, or substituted heteroaryl.

In another embodiment of the invention the compound of Formula I isselected from:

wherein:

R5, R6, R7, R8, R9, R11 and R12 are independently selected fromhydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

Compounds of the present invention include, but are not limited to thefollowing exemplary compounds:

The present invention includes pharmaceutically acceptable salts of thecompounds defined by Formula I. Compounds according to the presentinvention can possess a sufficiently acidic, a sufficiently basic, orboth functional groups, and accordingly react with a number of organicand inorganic bases, and organic and inorganic acids, to formpharmaceutically acceptable salts.

The term “pharmaceutically acceptable salt” as used herein, refers to asalt of a compound of Formula I, which is substantially non-toxic toliving organisms. Typical pharmaceutically acceptable salts includethose salts prepared by reaction of the compound of the presentinvention with a pharmaceutically acceptable mineral or organic acid oran organic or inorganic base. Such salts are known as acid addition andbase addition salts.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulphonic acid, methanesulphonic acid, oxalic acid,p-bromophenylsulphonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suchpharmaceutically acceptable salts are the sulphate, pyrosulphate,bisulphate, sulphite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate,xylenesulphonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,methanesulphonate, propanesulphonate, naphthalene-1-sulfonate,napththalene-2-sulfonate, mandelate and the like. Preferredpharmaceutically acceptable acid addition salts are those formed withmineral acids such as hydrochloric acid and hydrobromic acid, and thoseformed with organic acids such as maleic acid and methanesulphonic acid.

Salts of amine groups may also comprise quarternary ammonium salts inwhich the amino nitrogen carries a suitable organic group such as analkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, or aralkyl moiety.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing the salts of thisinvention thus include sodium hydroxide, potassium hydroxide, ammoniumhydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate,potassium bicarbonate, calcium hydroxide, calcium carbonate, and thelike.

One skilled in the art will understand that the particular counterionforming a part of a salt of this invention is usually not of a criticalnature, so long as the salt as a whole is pharmacologically acceptableand as long as the counterion does not contribute undesired qualities tothe salt as a whole. The present invention further encompasses thepharmaceutically acceptable solvates of a compound of Formula I. Many ofthe compounds of Formula I can combine with solvents such as water,methanol, ethanol and acetonitrile to form pharmaceutically acceptablesolvates such as the corresponding hydrate, methanolate, ethanolate andacetonitrilate.

The compounds of the present invention may have multiple asymmetric(chiral) centres. As a consequence of these chiral centres, thecompounds of the present invention occur as racemates, mixtures ofenantiomers and as individual enantiomers, as well as diastereomers andmixtures of diastereomers. All asymmetric forms, individual isomers andcombinations thereof, are within the scope of the present invention.

It will be readily understood by one skilled in the art that if thestereochemistry of a compound of Formula I is critical to its activity,then the relative stereochemistry of the compound is established earlyduring synthesis to avoid subsequent stereoisomer separation problems.Further manipulation of the molecule will then employ stereospecificprocedures so as to maintain the desired chirality.

Non-toxic metabolically-labile esters or amides of a compound of FormulaI are those that are hydrolysed in vivo to afford the compound ofFormula I and a pharmaceutically acceptable alcohol or amine. Examplesof metabolically-labile esters include esters formed with (1-6C)alkanols, in which the alkanol moiety may be optionally substituted by a(1-8C) alkoxy group, for example methanol, ethanol, propanol andmethoxyethanol. Non-limiting examples of metabolically-labile amidesinclude amides formed with amines such as methylamine.

II. Preparation of Compounds of Formula I

As is known in the art, triaryl imidazole compounds can be prepared by anumber of standard techniques. Compounds of Formula I, therefore, can beprepared by several general synthetic methods, for example, as describedby Grimmett, (Grimmett, M. R., Comprehensive Heterocyclic Chemistry: TheStructure, Reaction, Synthesis and Uses of Heterocyclic Compounds, A. R.Katrizky and C. W. Rees, eds., Vol. 5, Pergamon Press. Oxford, 1984, pp.457-498; Grimmett, M. R., Imidazole and Benzimidazole Synthesis,Academic Press, San Diego Calif., 1997).

In one embodiment of the present invention, compounds of Formula I areprepared via solution or solid phase synthesis, by reacting a dione ofFormula II with the aldehyde (III) at elevated temperature in thepresence of ammonium acetate in acetic acid (see, for example, Krieg etal., Naturforsch. 1967, 22b:132; Sarshar et al., Tetrahedron Lett. 1996,37:835-838).

The compounds of Formula (XXXI) and (XXXII) are either commerciallyavailable or may be prepared using standard procedures known to a personskilled in the relevant art. Compounds of Formula (XXXI), therefore, canbe prepared by several general synthetic methods, for example, asdescribed by: Fischer et. al (J. Am. Chem. Soc. 1961, 83, 4208-4210);Guijarro et al. (J. Am. Chem. Soc. 1999, 121, 4155-4157); Chi et. al.(Synth. Comm. 1994, 24(15), 2119-2122) and Armesto et. al. (Synthesis,1988, 799-801).

Compounds of formula XXXI can also be prepared:

-   -   i) by oxidizing a compound of formula (XXXII). Compounds of        formula (XXXIII), in turn can be prepared by reacting a        compounds of formula (XXXIV) with sodium cyanide in the presence        of a solvent as shown below, wherein R3=R2 and R2 is as defined        above:

or,

-   -   ii) by oxidizing a compound of formula (XXXV). Compounds of        formula (XXXV), in turn can be prepared by treating a compound        of formula (XXXIV) and a compound of formula (XXXVI) with sodium        cyanide in the presence of a solvent as shown below, wherein R2        and R3 are as defined above:

or,

-   -   iii) by oxidizing a compound of formula (XXXVII). Compounds of        formula (XXXVII) in turn can be prepared by oxidizing a compound        of formula (XXXVIII) or (XXXIX) as shown below, wherein R2 and        R3 are as defined above:

or,

-   -   iv) by oxidizing a compound of formula (XXXIX) using PdCl₂ in        DMSO,        or,    -   v) by deprotecting and oxidizing a compound of formula (XL).        Compounds of formula (XL) in turn can be prepared by reacting a        compound of formula (XLI) with a compound of formula (XLII) in        the presence of a suitable base:

-   -    wherein R2 and R3 are independently aryl, substituted aryl,        heteroaryl or substituted heteroaryl,        or,    -   vi) by reacting a compound of formula (XLIII) with a substituted        or unsubstituted aryl or substituted or unsubstituted heteroaryl        under Friedel-Crafts acylation conditions or by nucleophilic        displacement of the chloride in compound of formula (XLIII).        Compounds of formula (XLIII) in turn can be prepared by reacting        a substituted or unsubstituted aryl or substituted heteroaryl pr        unsubstituted heteroaryl with oxalyl chloride under        Friedel-Crafts acylation conditions:

-   -    wherein R2 and R3 are independently aryl, substituted aryl,        heteroaryl or substituted heteroaryl;        or    -   vii) by oxidising a compound of formula (XLIV). Compounds of        formula (XLIV) in turn can be prepared by reacting a compound of        formula (XLV) with thionyl chloride in benzene with catalytic        dimethylformamide to form an intermediate (XLVI). This        intermediate (XLVI) is then used directly without purification        in a Freidel-Crafts reaction to produce the ketone (XLIV).

III. Anti-cancer Activity of Compounds of Formula I

The ability of a candidate compound of Formula I to inhibit neoplasticcell growth and/or proliferation can be tested using standard techniquesknown in the art. In addition, compounds of Formula I that demonstrateinhibitory activity may be further tested in vitro and/or in vivo incombination with various known chemotherapeutics to evaluate theirpotential use in combination therapies. Exemplary methods of testingcandidate compounds of Formula I are provided below and in the Examplesincluded herein. One skilled in the art will understand that othermethods of testing the compounds are known in the art and are alsosuitable for testing candidate compounds.

A. In vitro Testing

Candidate compounds of Formula I can be assayed initially in vitro fortheir ability to inhibit cell growth (i.e. their cytotoxicity) usingstandard techniques. In general, cells of a specific test cell line(typically a cancer cell line) are grown to a suitable density (e.g.approximately 1×10⁴) and the candidate compound is added. After anappropriate incubation time (typically between about 48 to 74 hours),cell survival is assessed, for example, by assaying for tetrazolium salt(or modified tetrazolium salt) cleavage, or by using the resazurinreduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab.11:48-50; O'Brien et al., (2000) Eur J. Biochem. 267:5421-5426 and U.S.Pat. No. 5,501,959), the sulforhodamine assay (Rubinstein et al. (1990)J. Natl. Cancer Inst. 82:113-118) or the neutral red dye test (Kitano etal., (1991) Euro. J. Clin. Investg. 21:53-58; West et al., (1992) J.Investigative Derm. 99:95-100). Inhibition of cell growth is determinedby comparison of cell survival in the treated culture with cell survivalin one or more control cultures, for example, cultures not pre-treatedwith the candidate compound and/or those pre-treated with a controlcompound (typically, a known therapeutic). Other suitable techniques forassessing cytotoxicity are known in the art.

Assays that measure metabolic activity (such as tetrazolium-basedassays) can also be used to assess the effect of candidate compounds oncell activation and/or proliferation, due the fact that proliferatingcells are metabolically more active than resting cells.

Candidate compounds can also be tested in vitro for their ability toinhibit anchorage-independent growth of tumour cells.Anchorage-independent growth is known in the art to be a good indicatorof tumourigenicity. In general, anchorage-independent growth is assessedby plating cells from an appropriate cancer cell-line onto soft agar anddetermining the number of colonies formed after an appropriateincubation period. Growth of cells treated with the candidate compoundcan then be compared with that of cells treated with an appropriatecontrol (as described above).

A variety of cancer cell-lines suitable for testing the candidatecompounds are known in the art. In one embodiment of the presentinvention, in vitro testing of the candidate compounds is conducted in ahuman cancer cell-line. Examples of suitable human cancer cell-lines forin vitro testing of the compounds of the present invention include, butare not limited to, colon and colorectal carcinoma cell lines such asHT-29, CaCo, LoVo, COLO320 and HCT-116; non small cell lung cancer celllines such as NCI-H460, small cell lung cancer cell lines such as H209;breast cancer cell lines such as MCF-7, T47D and MDA-MB-231; ovariancancer cell lines such as SK-OV-3; prostate cancer cell lines such asPC-3 and DU-145; chronic myeloid leukaemia cell lines such as K562;bladder cancer cell lines such as T24; brain cancer cell lines such asU-87-MG; pancreatic cancer cell lines such as AsPC-1, SU.86.86 andBxPC-3; kidney cancer cell lines such as A498 and Caki-1; liver cancercell lines such as HepG2, and skin cancer cell lines such as A2058 andC8161. Drug-resistant cancer cell lines can be used to determine theability of the compounds of the present invention to inhibit growthand/or proliferation of drug- or multi-drug resistant neoplastic cells.

The selectivity of the candidate compounds of Formula I may also betested, i.e. the ability of the compound to demonstrate some level ofselective action toward neoplastic (or cancer) cells in comparison tonormal proliferating cells. An exemplary method of assessing thedifferential sensitivity between normal and cancer cells for a compoundhas been described by Vassilev et al. (Anti-Cancer Drug Design (2001)16:7). This method involves the comparison of IC90 values, i.e. themolar concentration of a test compound required to cause 90% growthinhibition of exponentially growing cells. Thus, the IC₉₀ values forcandidate compounds can be evaluated in various cancer cell lines (suchas those outlined above) and normal cells (such as HUVEC and/or WI38cells) and compared. IC₉₀ values can be measured using a variety ofstandard techniques including those described above for cytotoxicitytesting.

While the mechanism of action of the compounds of Formula I is notrelevant to the instant invention, assays to investigate potentialmechanisms of action of the compounds may be conducted if desired inorder to provide information useful in determining what aspects oftumour growth the compounds affect. This type of information may help todetermine cancer types that will benefit from treatment with thecompounds. Examples of such assays include, but are not limited to,cell-cycle analysis (for example, employing flow cytometry techniques),apoptosis assays (such as DNA fragmentation analysis), anti-angiogenesisassays (for example, various Matrigel assays, including cord formationand Matrigel plug assays) and immunohistochemical analysis.

Toxicity of the candidate compounds can also be initially assessed invitro using standard techniques. For example, human primary fibroblastscan be treated in vitro with a compound of Formula I and then tested atdifferent time points following treatment for their viability using astandard viability assay, such as the assays described above or thetrypan-blue exclusion assay. Cells can also be assayed for their abilityto synthesize DNA, for example, using a thymidine incorporation assay,and for changes in cell cycle dynamics, for example, using a standardcell sorting assay in conjunction with a fluorocytometer cell sorter(FACS).

B. In vivo Testing

The ability of the candidate compounds to inhibit tumour growth,proliferation and/or metastasis in vivo can be determined in anappropriate animal model using standard techniques known in the art(see, for example, Enna, et al., Current Protocols in Pharmacology, J.Wiley & Sons, Inc., New York, N.Y.). Exemplary protocols are providedbelow and in the Examples. Non-limiting examples of suitable animalmodels are provided in Table 1.

In general, current animal models for screening anti-tumour compoundsare xenograft models, in which a human tumour has been implanted into ananimal. For example, the candidate compounds can be tested in vivo onsolid tumours using mice that are subcutaneously grafted or injectedwith 30 to 60 mg of a tumour fragment, or an appropriate number oftumour cells (e.g. about 10⁶ to 10⁷) on day 0. The animals bearingtumours are mixed before being subjected to the various treatments andcontrols. In the case of treatment of advanced tumours, tumours areallowed to develop to the desired size, animals having insufficientlydeveloped tumours being eliminated. The selected animals are distributedat random to undergo the treatments and controls. Animals not bearingtumours may also be subjected to the same treatments as thetumour-bearing animals in order to be able to dissociate the toxiceffect from the specific effect on the tumour. Chemotherapy generallybegins from 3 to 22 days after grafting, depending on the type oftumour, and the animals are observed every day. Candidate compounds canbe administered to the animals, for example, by bolus infusion. Thedifferent animal groups are weighed about 3 or 4 times a week until themaximum weight loss is attained, after which the groups are weighed atleast once a week until the end of the trial. The tumours are measuredabout 2 or 3 times a week until the tumour reaches a pre-determined sizeand/or weight, or until a pre-determined time period has passed, oruntil the animal dies (if this occurs before the tumour reaches thepre-determined size/weight). The animals are then sacrificed and thetissue histology, size and/or proliferation of the tumour assessed.

If desired, one or more standard immunohistochemical tests may also beconducted on tissues isolated from the test animals in order todetermine the effects of the compound on tumour growth, differentiation,apoptosis and/or angiogenesis. Examples of such tests include, but arenot limited to, the use of specific antibodies (for example, antibodiesagainst Ki-67 to assess proliferation, CD31 to assess angiogenesis,NK1.1 as an indication of the presence of NK cells, F4/80 as anindication of the presence of macrophages) and TUNEL assays to determineapoptosis. Other models, such as orthopedic implantation of tumours intoanimals (i.e. the implantation of cancer cells of a certain type intothe corresponding tissue in the animal, such as pancreatic cancer cellsinto the pancreas), may also be used to assess the effect of thecandidate compounds on tumour growth and proliferation. In addition, theeffect of the candidate compound on spontaneous tumours in normal micecan be assessed.

The effect of the candidate compounds on drug-resistant tumours can beassessed in vivo by utilising a drug- or multidrug-resistant cancer cellin the xenograft experiments.

For the study of the effect of the candidate compounds on haematologictumours, such as lymphomas or leukaemias, the animals are grafted orinjected with a particular number of cells, and the anti-tumour activityis determined by the increase in the survival time of the treated micerelative to the controls.

To study the effect of the candidate compounds on tumour metastasis,tumour cells are typically treated with the compound ex vivo and theninjected into a suitable test animal. The spread of the tumour cellsfrom the site of injection is then monitored over a suitable period oftime.

The ability of the candidate compounds to act in combination with, or tosensitise a tumour to the effects of, another chemotherapeutic agent canalso be tested in the above models. In this case, the test animals wouldbe treated with both the chemotherapeutic agent and the candidatecompound of Formula I. Control animals could include animals treatedwith the chemotherapeutic alone, animals treated with the candidatecompound alone and/or untreated animals.

In vivo toxic effects of the compounds of Formula I can be evaluated bystandard techniques, for example, by measuring their effect on animalbody weight during treatment and by performing haematological profilesand liver enzyme analysis after the animal has been sacrificed (survivalassays).

TABLE I Examples of in vivo models of human cancer Cancer Model CellType Tumour Growth Assay Prostate (PC-3, DU145) Human solid tumourxenografts in Breast (MDA-MB-231, MVB-9) mice (sub-cutaneous injection)Colon (HT-29) Lung (NCI-H460, NCI-H209) Pancreatic (ASPC-1, SU86.86)Pancreatic: drug resistant (BxPC-3) Skin (A2058, C8161) Cervical (SIHA,HeLa-S3) Cervical: drug resistant (HeLa S3-HU-resistance) Liver (HepG2)Brain (U87-MG) Renal (Caki-1, A498) Ovary (SK-OV-3) Bladder (T24) TumourGrowth Assay Breast: drug resistant (MDA-CDDP- Human solid tumourisografts in S4, MDA-MB435-To.1) mice (fat pad injection) Survival AssayHuman: Burkitts lymphoma (Non- Experimental model of lymphoma Hodgkin's)(raji) and leukaemia in mice Murine: erythroleukemia (CB7 Friendretrovirus-induced) Experimental model of lung. Human: melanoma (C8161)metastasis in mice Murine: fibrosarcoma (R3)

IV. Toxicity Testing

It is important that the anti-cancer compounds of the present inventionexhibit low toxicity in vivo. Toxicity tests for potential drugs arewell-known in the art (see, for example, Hayes, A. W., ed., (1994),Principles and Methods of Toxicology, 3^(rd) ed., Raven Press, NY;Maines, M., ed., Current Protocols in Toxicology, John Wiley & Sons,Inc., NY).

In vitro acute toxicity testing of a compound of Formula I can beperformed using mammalian cell, lines (see, for example, Ekwall, B.,Ann. N.Y. Acad. Sci., (1983) 407:64-77). Selection of an appropriatecell line is dependent on the potential application of the candidatecompound and can be readily determined by one skilled in the art.

In vivo toxicity testing can be performed by standard methodology. Forexample, by injecting varying concentrations of the candidate compoundinto an appropriate animal model. The compound can be injected once, oradministration can be repeated over several days. The toxic effects ofthe compound can be evaluated over an appropriate time period bymonitoring the general health and body weight of the animals. After thecompletion of the period of assessment, the animals can be sacrificedand the appearance and weight of the relevant organs determined. Anindication of the toxicity of a compound can also be obtained during thein vivo anti-cancer testing of the compound.

V. Therapeutic Uses of Compounds of Formula I

The compounds of Formula I can be used in the treatment and/orstabilisation of various types of cancers. In this context, thecompounds may exert either a cytotoxic or cytostatic effect resulting ina reduction in the size of a tumour, the slowing or prevention of anincrease in the size of a tumour, an increase in the disease-freesurvival time between the disappearance or removal of a tumour and itsreappearance, prevention of an initial or subsequent occurrence of atumour (e.g. metastasis), an increase in the time to progression,reduction of one or more adverse symptom associated with a tumour, or anincrease in the overall survival time of a subject having cancer. Thecompounds can be used alone or they can be used as part of a multi-drugregimen in combination with one or more known therapeutics.

Examples of cancers which may be may be treated or stabilized inaccordance with the present invention include, but are not limited tohaematologic neoplasms, including leukaemias and lymphomas; carcinomas,including adenocarcinomas; melanomas and sarcomas. Carcinomas,adenocarcinomas and sarcomas are also frequently referred to as “solidtumours.” Examples of commonly occurring solid tumours include, but arenot limited to, cancer of the brain, breast, cervix, colon, head andneck, kidney, lung, ovary, pancreas, prostate, stomach and uterus,non-small cell lung cancer and colorectal cancer. Various forms oflymphoma also may result in the formation of a solid tumour and,therefore, are also often considered to be solid tumours. One embodimentof the present invention provides for the use of the compounds ofFormula I in the treatment and/or stabilisation of a solid tumour.

The term “leukaemia” refers broadly to progressive, malignant diseasesof the blood-forming organs. Leukaemia is typically characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow but can also refer to malignantdiseases of other blood cells such as erythroleukaemia, which affectsimmature red blood cells. Leukaemia is generally clinically classifiedon the basis of (1) the duration and character of the disease—acute orchronic; (2) the type of cell involved—myeloid (myelogenous), lymphoid(lymphogenous) or monocytic, and (3) the increase or non-increase in thenumber of abnormal cells in the blood—leukaemic or aleukaemic(subleukaemic). Leukaemia includes, for example, acute nonlymphocyticleukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia,chronic granulocytic leukaemia, acute promyelocytic leukaemia, adultT-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia,basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronicmyelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilicleukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblasticleukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cellleukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphaticleukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenousleukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cellleukaemia, megakaryocyte leukaemia, micromyeloblastic leukaemia,monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia,myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegelileukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocyticleukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cellleukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia.

The term “sarcoma” generally refers to a tumour which originates inconnective tissue, such as muscle, bone, cartilage or fat, and is madeup of a substance like embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include soft tissue sarcomas, chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented haemorrhagic sarcoma, immunoblasticsarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumour arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma, subungal melanoma, and superficial spreadingmelanoma.

The terra “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma,epiermoid carcinoma, carcinoma epitheliale adenoides, exophyticcarcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniformcarcinoma, gelatinous carcinoma, giant cell carcinoma, carcinomagigantocellulare, glandular carcinoma, granulosa cell carcinoma,hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma,Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma,infantile embryonal carcinoma, carcinoma in situ, intraepidermalcarcinoma, intraepithelial carcinoma, Krompecher's carcinoma,Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma,carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma,carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinomamolle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare,mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinomamyxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cellcarcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma,periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma,pultaceous carcinoma, renal cell carcinoma of kidney, reserve cellcarcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhouscarcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinomasimplex, small-cell carcinoma, solanoid carcinoma, spheroidal cellcarcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

The term “carcinoma” also encompasses adenocarcinomas. Adenocarcinomasare carcinomas that originate in cells that make organs which haveglandular (secretory) properties or that originate in cells that linehollow viscera, such as the gastrointestinal tract or bronchialepithelia. Examples include, but are not limited to, adenocarcinomas ofthe breast, lung, colon, pancreas and prostate.

Additional cancers encompassed by the present invention include, forexample, Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma,neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumours, primary brain tumours,malignant pancreatic insulanoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, gliomas, testicular cancer, thyroidcancer, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, mesotheliomaand medulloblastoma.

The cancer to be treated or stabilized may be indolent or it may beaggressive. The compounds of the invention can be used to treatrefractory cancers, advanced cancers, recurrent cancers and metastaticcancers. One skilled in the art will appreciate that many of thesecategories may overlap, for example, aggressive cancers are typicallyalso metastatic.

“Aggressive cancer,” as used herein, refers to a rapidly growing cancer.One skilled in the art will appreciate that for some cancers, such asbreast cancer or prostate cancer the term “aggressive cancer” will referto an advanced cancer that has relapsed within approximately the earliertwo-thirds of the spectrum of relapse times for a given cancer, whereasfor other types of cancer, such as small cell lung carcinoma (SCLC),nearly all cases present rapidly growing cancers which are considered tobe aggressive. The term can thus cover a subsection of a certain cancertype or it may encompass all of other cancer types. A “refractory”cancer or tumour refers to a cancer or tumour that has not responded totreatment. “Advanced cancer,” refers to overt disease in a patient thatis not amenable to cure by local modalities of treatment, such assurgery or radiotherapy. Advanced disease may refer to a locallyadvanced cancer or it may refer to metastatic cancer. The term“metastatic cancer” refers to cancer that has spread from one part ofthe body to another.

The present invention also contemplates the use of the compounds ofFormula I as “sensitizing agents,” which selectively inhibit the growthof cancer cells. In this case, the compound alone does not have acytotoxic effect on the cancer cell, but provides a means of weakeningthe cancer cells, and thereby facilitates the benefit from conventionalanti-cancer therapeutics.

Thus, the present invention contemplates the administration to a subjectof a therapeutically effective amount of one or more compound of FormulaI together with one or more anti-cancer therapeutics. The compound(s)can be administered before, during or after treatment with theanti-cancer therapeutic. An “anti-cancer therapeutic” is a compound,composition or treatment that prevents or delays the growth and/ormetastasis of cancer cells. Such anti-cancer therapeutics include, butare not limited to, chemotherapeutic drug treatment, radiation, genetherapy, hormonal manipulation, immunotherapy and antisenseoligonucleotide therapy. A wide variety of chemotherapeutic drugs areknown in the art and can be used in combination therapies with acompound of the present invention. Examples of useful chemotherapeuticdrugs include broad spectrum chemotherapeutics, i.e. those that areuseful in the treatment of a range of cancers, such as doxorubicin,capecitabine, mitoxantrone, irinotecan (CPT-11), cisplatin andgemcitabine. Other examples of useful chemotherapeutic agents include,but are not limited to, hydroxyurea, busulphan, carboplatin,chlorambucil, melphalan, cyclophosphamide, Ifosphamide, danorubicin,epirubicin, vincristine, vinblastine, Navelbine® (vinorelbine),etoposide, teniposide, paclitaxel, docetaxel, cytosine, arabinoside,bleomycin, neocarcinostatin, suramin, taxol, mitomycin C and the like.The compounds of the invention are also suitable for use with standardcombination therapies employing two or more chemotherapeutic agents. Itis to be understood that anti-cancer therapeutics for use in the presentinvention also include novel compounds or treatments developed in thefuture.

VI. Pharmaceutical Compositions

The compounds of the present invention are typically formulated prior toadministration. The present invention thus provides pharmaceuticalcompositions comprising one or more compounds of Formula I and apharmaceutically acceptable carrier, diluent, or excipient. Thepharmaceutical compositions are prepared by known procedures usingwell-known and readily available ingredients. Pharmaceuticalcompositions comprising one or mare compounds of Formula I incombination with one or more known cancer chemotherapeutics are alsocontemplated by the present invention.

Compounds of the general Formula I or pharmaceutical compositionscomprising the compounds may be administered orally, topically,parenterally, by inhalation or spray, or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. In the usual course oftherapy, the active compound is incorporated into an acceptable vehicleto form a composition for topical administration to the affected area,such as hydropohobic or hydrophilic creams or lotions, or into a formsuitable for oral, rectal or parenteral administration, such as syrups,elixirs, tablets, troches, lozenges, hard or soft capsules, pills,suppositiories, oily or aqueous suspensions, dispersible powders orgranules, emulsions, injectables, or solutions. The term parenteral asused herein includes subcutaneous injections, intradermal,intra-articular, intravenous, intramuscular, intravascular,intrasternal, intrathecal injection or infusion techniques.

Compositions intended for oral use may be prepared in either solid orfluid unit dosage forms. Fluid unit dosage form can be preparedaccording to procedures known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavouring agents, colouring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. An elixiris prepared by using a hydroalcoholic (e.g., ethanol) vehicle withsuitable sweeteners such as sugar and saccharin, together with anaromatic flavoring agent. Suspensions can be prepared with an aqueousvehicle with the aid of a suspending agent such as acacia, tragacanth,methylcellulose and the like.

Solid formulations such as tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents for example, corn starch, or alginic acid; bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc and otherconventional ingredients such as dicalcium phosphate, magnesium aluminumsilicate, calcium sulfate, starch, lactose, methylcellulose, andfunctionally similar materials. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate maybe employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil. Softgelatin capsules are prepared by machine encapsulation of a slurry ofthe compound with an acceptable vegetable oil, light liquid petrolatumor other inert oil.

Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl-p-hydroxy benzoate, one or more colouringagents, one or more flavouring agents or one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example peanut oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil, forexample olive oil or peanut oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor a suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Adjuvants such as local anaesthetics,preservatives and buffering agents can also be included in theinjectable solution or suspension.

The compound(s) of the general Formula I may be administered, togetheror separately, in the form of suppositories for rectal administration ofthe drug. These compositions can be prepared by mixing the drug with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials include cocoabutter and polyethylene glycols.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

VII. Administration of Compounds of Formula I

Compounds of Formula I may be administered to a subject by a variety ofroutes depending on the cancer to be treated, for example, the compoundsmay be administered orally, topically, parenterally, by inhalation orspray, or rectally in dosage unit formulations. In one embodiment, thecompounds are administered systemically to a subject, for example, bybolus injection or continuous infusion into a subject's bloodstream orby oral administration. When used in conjunction with one or more knownchemotherapeutic agents, the compounds can be administered prior to, orafter, administration of the chemotherapeutic agents, or they can beadministered concomitantly. The one or more chemotherapeutic may also beadministered systemically, for example, by bolus injection, continuousinfusion, or oral administration.

The compounds of Formula I may be used as part of a neo-adjuvant therapy(to primary therapy), or as part of an adjuvant therapy regimen, wherethe intention is to cure the cancer in a subject. The present inventioncontemplates the use of the compounds of Formula I at various stages intumour development and progression, including in the treatment ofadvanced and/or aggressive neoplasias (i.e. overt disease in a subjectthat is not amenable to cure by local modalities of treatment, such assurgery or radiotherapy), metastatic disease, locally advanced diseaseand/or refractory tumours (i.e. a cancer or tumour that has notresponded to treatment).

“Primary therapy” refers to a first line of treatment upon the initialdiagnosis of cancer in a subject. Exemplary primary therapies mayinvolve surgery, a wide range of chemotherapies and radiotherapy.“Adjuvant therapy” refers to a therapy that follows a primary therapyand that is administered to subjects at risk of relapsing. Adjuvantsystemic therapy is usually begun soon after primary therapy to delayrecurrence, prolong survival or cure a subject.

It is contemplated that the compounds of the invention can be used aloneor in combination with one or more other chemotherapeutic agents as partof a primary therapy or an adjuvant therapy. Combinations of thecompounds of Formula I and standard chemotherapeutics may act to improvethe efficacy of the chemotherapeutic and, therefore, can be used toimprove standard cancer therapies. This application can be important inthe treatment of drug-resistant cancers which are not responsive tostandard treatment. Drug-resistant cancers can arise, for example, fromheterogeneity of tumour cell populations, alterations in response tochemotherapy and increased malignant potential. Such changes are oftenmore pronounced at advanced stages of disease.

The dosage to be administered is not subject to defined limits, but itwill usually be an effective amount. It will usually be the equivalent,on a molar basis of the pharmacologically active free form produced froma dosage formulation upon the metabolic release of the active free drugto achieve its desired pharmacological and physiological effects. Thecompositions may be formulated in a unit dosage form. The term “unitdosage form” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient. Examples of ranges for the compound(s) in eachdosage unit are from about 0.05 to about 100 mg, or more usually, fromabout 1.0 to about 50 mg.

Daily dosages of the compounds of the present invention will typicallyfall within the range of about 0.01 to about 100 mg/kg of body weight,in single or divided dose. However, it will be understood that theactual amount of the compound(s) to be administered will be determinedby a physician, in the light of the relevant circumstances, includingthe condition to be treated, the chosen route of administration, theactual compound administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms. Theabove dosage range is given by way of example only and is not intendedto limit the scope of the invention in any way. In some instances dosagelevels below the lower limit of the aforesaid range may be more thanadequate, while in other cases still larger doses may be employedwithout causing harmful side effects, for example, by first dividing thelarger dose into several smaller doses for administration throughout theday.

VIII. Kits

The present invention additionally provides for therapeutic kitscontaining one or more compounds of Formula I for use in the treatmentof cancer. The contents of the kit can be lyophilized and the kit canadditionally contain a suitable solvent for reconstitution of thelyophilized components. Individual components of the kit would bepackaged in separate containers and, associated with such containers,can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,for use or sale for human or animal administration.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. For in vivo use, the compounds may beformulated into a pharmaceutically acceptable syringeable composition.In this case the container means may itself be an inhalant, syringe,pipette, eye dropper, or other such like apparatus, from which theformulation may he applied to an infected area of the subject, such asthe lungs, injected into an subject, or even applied to and mixed withthe other components of the kit.

Pharmaceutical kits or packs comprising one or more compound of thepresent invention in combination with one or more standardchemotherapeutic for combination therapy applications are alsocontemplated by the present invention. To gain a better understanding ofthe invention described herein, the following examples are set forth. Itshould be understood that these examples are for illustrative purposesonly. Therefore, they should not limit the scope of this invention inany way.

EXAMPLES Preparation of Compounds

All reactions have been carried out according to the scheme shown below:

In a typical experimental procedure 1 mmol (1 equiv.) of thecarboxyaldehyde was combined with 1.05-1.10 mmole (1.05-1.1 equiv.) ofthe dione and 20 mmole (20 equiv.) of ammonium acetate and 5 ml ofacetic acid. The mixture was magnetically stirred and heated to refluxfor 3-5 hr. The reaction process was monitored by TLC, until completeconsumption of the indole was achieved. The reaction mixture was cooledto room temperature and added drop-wise into well-stirred ice-water. Thesuspension solid was then filtered and the crude solid was dissolved inethyl acetate, dried over sodium sulfate and filtered, the organicsolvent was removed by vacuum. The products was then eitherrecrystalized with alcohol or separated by column chromatography usingpetroleum ether-Ethyl acetate as an eluant.

Melting points were recorded using a MEL-TEMP capillary melting pointapparatus, the melting point are uncorrected. ¹H-NMR was performed in a500 MHz Brucker instrument at room temperature using a suitabledeuterated solvent.

Example 1 Preparation of Compound 2

1 mmol (1 equiv.) of the indole carboxyaldehyde was combined with1.05-1.10 mmole (1.05-1.1 equiv.) of the benzil and 20 mmole (20 equiv.)of ammonium acetate and 5 ml of acetic acid. The mixture wasmagnetically stirred and heated to reflux for 3-5 hr. The reactionprocess was monitored by TLC, until complete consumption of the indolewas achieved. The reaction mixture was cooled to room temperature andadded drop-wise into well-stirred ice-water. The suspension solid wasthen filtered and the crude solid was dissolved in ethyl acetate, driedover sodium sulfate and filtered, the organic solvent was removed byvacuum. The products was then either recrystalized with alcohol orseparated by column chromatography using petroleum ether-Ethyl acetateas an eluant.

It is noteworthy that, the TLC of the products shows a characteristicblue florescent color under the UV (Wave length λ=254 nm), a propertyused as an additional characterization feature.

¹H-NMR: δ (DMSO-d₆), 12.10 (s, 1H), 11.30 (s, 1H), 7.98 (d, 1H), 7.62(d, 2H), 7.56 (d, 2H), 7.45 (t, 2H), 7.28-7.40 (m, 4H), 7,24 (t, 1H),7.03-7.14 (m, 2H), 2.70 (s, 3H). HRMS m/z for C₂₄H₁₉N₃ calc. Is349.157898, found 349.157897. M.p.=decomposed at 260-264.

The following exemplary compounds were also prepared from theappropriate starting materials following the general synthetic procedureas discussed above.

Example 2 Compound 5

¹H-NMR (CDCl₃): δ=8.02 (d, 2H), 7.53 (d, 1H), 7.43-7.52 (m, 6H), 7.41(d, 1H), 7.21-7.34 (m, 6H), 2.81 (s, 3H), 2.75 (s, 3H). EIMS [M⁺] m/zfor C₂₈H₂₃N₃O₂ is 433. M.p.=224-227.

Example 3 Compound 10

¹H-NMR (CDCl₃): δ=10.68 (bs, 1H), 7.73 (bs, 1H), 7.22 (d, 4H), 6.99 (bs,1H), 6.92 (bd, 2H), 6.85 (bd, 2H), 6.611 (d, 4H), 3.70 (s, 6H). EIMS[M⁺] m/z for C₂₅H₂₀N₃BrO₂ is 474. M.p.=135.

Example 4 Compound 11

¹H-NMR (CDCl₃): δ=7.70 (d, 1H), 7.41 (d, 4H), 7.32 (d, 1H), 7.09 (q,2H), 6.77 (d, 4H), 2.95 (s, 12H), 2.67 (s, 3H). EIMS [M⁺] m/z forC₂₈H₂₉N₅ is 435, M.p.=decomposed at 236-238.

Example 5 Compound 13

¹H-NMR (CDCl₃): δ=7.47 (d, 4H), 7.44 (d, 4H), 7.30-7.34 (m, 1H),7.14-7.19 (m, 3H), 2.68 (bs, 3H), EIMS [M⁺] m/z for C₂₄H₁₇N₃Br₂ is 507.M.p.=240-245.

Example 6 Compound 19

¹H-NMR (DMSO-d₆): δ=12.13 (s, 1H), 11.33 (s, 1H), 7.94 (d, 2H), 7.57 (d,2H), 7.52 (bd, 2H), 7.39 (bd, 2H), 7.35 (d, 1H), 7.05-7.12 (m, 3H), 2.50(s, 3H). EIMS [M⁺] m/z for C₂₄H₁₇N₃Cl₂ is 418. M.p.=165-167.

Example 7 Compound 22

¹H-NMR (DMSO-d₆): δ=13.176 (s) 1H, 12.130 (s) 1H, 8.996 (d) 1H, 8.889(d) 1H, 8.852 (d) 1H, 8.671 (d) 1H 8.412 (d) 1H, 8.378 (d) 1H,7.775-7.750 (m) 2H, 7.640-7.600 (m) 2H.

Example 8 Compound 26

¹H-NMR (DMSO-d₆): δ=12.60 (s, 1H), 11.70 (s, 1H), 8.60 (d, 1H), 8.17 (s,1H), 7.68 (bs, 1H), 7.46 (d, 2H), 7.33 (d, 2H), 7.25 (bs, 2H), 7.09 (bs,1H). EIMS [M⁺] m/z for C₁₉H₁₂N₃BrS₂ is 426.

Example 9 Compound 28

¹H-NMR (DMSO-d₆): δ=12.60 (s, 1H), 11.65 (s, 1H), 8.44-8.64 (m, 3H),8.01-8.14 (m, 1H), 7.22-7.66 (m, 8H). EIMS [M⁺] m/z for C₂₂H₁₄N₄BrF is433. M.p.=decomposed at 343.

Example 10 Compound 29

¹H-NMR (DMSO-d₆): δ=8.83 (q, 2H), 8.73 (m, 1H), 8.68 (d, 1H), 8.46 (d,1H), 8.24 (s, 1H), 7.74 (t, 2H), 7.62 (t, 2H), 7.51-7.56 (m, 1H),7.23-7.27 (m, 2H), 2.71 (s, 3H). EIMS [M⁺] m/z for C₂₃H₁₅N₃ is 303.M.p.=135-137.

Example 11 Compound 31

¹H-NMR (CDCl₃): δ=8.90 (bs, 1H), 7.62 (bs, 1H), 7.48 (bd, 4H), 7.34 (m,4H), 7.21 (m, 1H), 7.13 (m, 2H), 2.43 (bs, 3H). EIMS [M⁺] m/z forC₂₃H₁₅N₃ClBr is 448. M.p.=decomposed at 218-220.

Example 12 Compound 32

¹H-NMR (CDCl₃): δ=8.12 (bs, 1H), 7.48 (d, 2H), 7.46 (d, 2H), 7.23-7.34(m, 8H). M.p.=230-232.

Example 13 Compound 34

¹H-NMR (DMSO-d₆): δ=12.63 (s, 1H), 11.67 (s, 1H), 8.62 (d, 1H), 8.21 (d,2H), 8.08 (d, 1H), 7.86 (d, 2H), 7.39-7.64 (m, 6H), 7.32 (dd, 1H). EIMS[M⁺] m/z for C₂₃H₁₅N₄BrFO₂ is 459. M.p.=decomposed at 250-253.

Example 14 Compound 35

¹H-NMR (CDCl₃): δ=7.78 (bs, 1H), 7.59 (d, 2H), 7.54 (d, 2H), 7.35-7.39(m, 2H), 7.28-7.34 (m, 2H), 7.13-7.18 (m, 2H), 7.01-7.05 (m, 2H), 2.72(bs, 3H). EIMS [M⁺] m/z for C₂₄H₁₈N₃F is 367. M.p.=decomposed at247-250.

Example 15 Compound 36

¹H-NMR (CDCl₃): δ=10.42 (bs, 1H), 7.86 (s, 1H), 7.16-7.33 (m, 6H), 7.04(dd, 2H), 6.95 (dd, 2H), 6.88 (t, 3H). EIMS [M⁺] m/z for C₂₃H₁₅N₃BrF is432. M.p.=decomposed at 83-86.

Example 16 Compound 37

¹H-NMR (CDCl₃): δ=9.92 (bs, 1H), 8.17 (bs, 1H), 7.87 (t, 1H), 7.55 (bs,1H), 7.21-7.33 (m, 6H), 7.15-7.2 (m, 1H), 7.04-7.07 (m, 2H), 6.90 (t,2H)). EIMS [M⁺] m/z for C₂₃H₁₆N₃F is 353. M.p.=51.

Example 17 Compound 38

¹H-NMR (Acetone-d₆): δ=11.12 (bs, 1H), 10.46 (bs, 1H), 8.12 (d, 1H),7.80 (bd, 2H), 7.62 (bd, 2H), 7.38-7.48 (m, 5H), 6.98-7.22 (m, 12H),2.84 (bs, 3H). EIMS [M⁺] m/z for C₃₆H₂₇N₃O₂ is 533. M.p.=decomposed at128-130.

Example 18 Compound 40

¹H-NMR (CDCl₃): δ=8.12 (dd, 2H), 7.60 (m, 6H), 7.24-7.53 (m, 10H), 6.87(bd, 2H), 6.61 (bd, 2H), 2.08 (s, 3H). M.p.=decomposed at 142.

Example 19 Compound 41

¹H-NMR (CDCl₃): δ=8.08 (d, 4H), 8.07 (bs, 1H), 7.75 (d, 4H), 7.28-7.50(m, 10H), 7.12 (bd, 2H), 6.97 (bs, 1H). M.p.=155-158.

Example 20 Compound 42

¹H-NMR (CDCl₃): δ=9.39 (bs, 1H), 7.39-7.50 (m, 4H), 7.28-7.38 (m, 6H),7.06 (bs, 1H), 6.94 (bs, 2H), 2.08 (bs, 3H). EIMS [M⁺] m/z forC₂₄H₁₈N₃Br is 428. M.p.=decomposed at 155-157.

Example 21 Compound 43

¹H-NMR (CDCl₃): δ=9.75 (bs, 1H), 7.83 (bs, 1H), 7.36 (m, 3H), 7.25-7.29(m, 5H), 7.12 (m, 3H), 7.10 (bd, 1H). EIMS [M⁺] m/z for C₂₃H₁₅N₃Br₂ is493. M.p.=decomposed at 230.

Example 22 Compound 44

¹H-NMR (CDCl₃): δ=8.78 (dd, 2H), 8.19 (dd, 1H), 7.96 (bs, 1H), 7.80 (dd,1H), 7.80 (dd, 1H), 7.55-7.77 (m, 6H), 7.16-7.42 (m, 2H), 2.87 (bs, 3H).EIMS [M⁺] m/z for C₂₄H₁₇N₃ is 347. M.p.=decomposed at 167.

Example 23 Compound 45

¹H-NMR (DMSO-d₆): δ=13.30 (bs, 1H), 11.62 (d, 1H), 8.87 (bd, 2H), 8.64(bs, 1H), 8.44 (bs, 1H), 8.29 (t, 1H), 7.76 (t, 2H), 7.62 (t, 2H), 7.52(d, 1H), 7.35-7.41 (m, 2H). EIMS [M⁺] m/z for C₂₃H₁₄N₃Br is 412.

Example 24 Compound 46

¹H-NMR (DMSO-d₆): δ=13.09 (s, 1H), 11.61 (d, 1H), 8.83 (q, 2H), 8.73 (m,1H), 8.68 (d, 1H), 8.46 (d, 1H), 8.24 (s, 1H), 7.74 (t, 2H), 7.62 (t,2H), 7.51-7.56 (m, 1H), 7.23-7.27 (m, 2H). EIMS [M⁺] m/z for C₂₃H₁₅N₃ is333. M.p.=135-137.

Example 25 Compound 74

¹H-NMR (DMSO-d₆): δ=11.74 (d, 1H), 7.95 (dd, 1H), 7.32-7.57 (m, 6H),7.17-7.31 (m, 2H), 7.12 (t, 1H), 6.70 (d, 1H), 6.66 (bd, 1H). EIMS [M⁺]m/z for C₂₃H₁₉N₂FO₂ is 374.

Example 26 Compound 83

¹H-NMR (DMSO-d₆): δ=12.40 (s, 1H), 11.80 (s, 1H), 8.56 (d, 1H), 8.24 (s,1H), 8.15 (s, 1H), 7.78 (d, 1H), 7.65 (d, 2H), 7.54 (d, 2H), 7.47 (t,2H), 7.32-7.42 (m, 3H), 7.24 (t, 1H), 3.90 (s, 3H). EIMS [M⁺] m/z forC₂₅H₁₉N₃O₂ is 393. M.p.=293-295.

Example 27 Compound 84

¹H-NMR (DMSO-d₆): δ=8.64 (d, 1H), 8.17 (d, 1H), 7.47 (d, 1H), 7.39 (t,1H), 7.33 (dd, 1H), 7.20-7.31 (m, 2H), 7.12 (bd, 2H), 6.97 (bd, 1H),6.84 (bd, 1H). 3.77 (s, 3H), 3.72 (s, 3H), EIMS [M⁺] m/z forC₂₅H₂₀N₃BrO₂ is 474. M.p.=decomposed at 250-253.

Example 28 Compound 88

Mp 335-336° C. ¹H-NMR (DMSO-d₆), two isomers: 1) δ=13.160 (s) 1H, 11.602(s) 1H, 9.720 (s) 1H, 9.143 (dd) 1H, 8.975 (dd) 1H, 8.680 (d) 1H, 8.345(t) 1H, 8.160 (d) 1H, 7.870 (t) 1H, 7.720 (t) 1H, 7.420 (d) 1H, 7.200(d) 2H, 2.862 (s) 3H.

2): δ=13.090 (s) 1H, 11.602 (s) 1H, 9.370 (s) 1H, 9.143 (dd) 1H, 8.975(dd) 1H, 8.680 (d) 1H, 8.345 (t) 1H, 8.099 (d) 1H, 7.870 (t) 1H, 7.720(t) 1H, 7.420 (d) 1H, 7.200 (d) 2H, 2.847 (s) 3H. EI-MS(C₂₄H₁₆N₄O₂)=392.

Example 29 Compound 90

¹H-NMR (DMSO-d₆): δ=13.083 (s) 1H, 11.595 (s) 1H, 9.040-9.010 (m) 4H,8.950 (d) 1H, 8.120 (m) 1H, 7.821 (t) 1H, 7.432 (m) 1H, 7.176 (m) 2H,2.830 (s) 3H.

Example 30 Compound 92

¹H-NMR (DMSO-d₆): δ=12.729 (s) 1H, 11.724 (s) 1H, 8.578 (d) 1H, 8.325(d) 2H, 8.260 (d) 2H, 8.127 (d) 1H, 7.871 (m) 2H, 7.810-7.785 (m) 2H,7.454 (d) 1H, 7.330 (d) 1H.

Example 31 Compound 94

Mp 300-303° C. ¹H-NMR (DMSO-d₆): δ=12.55 (s) 1H, 8.83 (m) 3H, 8.68 (m)1H, 8.50 (m) 1H, 8.15 (m) 1H, 7.75 (m) 2H, 7.65 (m) 1H, 7.47 (m) 1H,7.40 (m) 1H. ESI-MS (C₂₃H₁₅BrN₄)=427.

Example 32 Compound 96

Mp 265-266° C. ¹H-NMR (DMSO-d₆): δ=12.1 (s) 1H, 11.6 (s) 1H, 8.7 (d) 1H,8.0 (d) 1H, 7.3 (m) 10H, 3.8 (d) 3H, 3.6 (d) 3H. ESI-MS(C₂₅H₂₀BrN₃O₂)=474.

Example 33 Compound 97

¹H-NMR (DMSO-d₆): δ=13.460 (s) 1H, 11.890 (s) 1H, 9.080-8.985 (m) 4H,8.860-8.825 (m) 1H, 8.285 (d) 1H, 7.890-7.840 (m) 2H, 7.560-7.540 (m)1H, 7.420-7.390 (m) 1H. ESI-MS (C₂₁H₁₂BrN₅)=414.

Example 34 Compound 101

EI-MS: 484.00 (C₂₅H₁₈BrN₅O requires 483.07) H¹ NMR (DMSO-d₆) d=13.167(s) 1H, 11.845 (s) 1H, 8.839-8.781 (m) 4H, 8.292-8.405 (m) 2H,7.364-7.697 (m) 5H, 3.543 (s) 1H, 3.410 (s) 2H, 1.463 (s) 2H.

Example 35 Compound 140

Mp 210-215° C. ¹H-NMR (DMSO-d₆): δ=12.18 (s) 1H, 11.54 (s) 1H, 9.08 (d)1H, 8.62 (s) 1H, 8.65 (s) 1H, 7.20 (m) 10H.

Example 36 Compound 141

¹H-NMR (DMSO-d₆): δ=12.30 (s) 1H, 11.70 (s) 1H, 7.79 (m) 3H, 7.58 (m)2H, 7.43 (m) 7H, 7.32 (m) 2H, 7.23 (m) 2H, 7.14 (m) 2H.

Example 37 Compound 146

Mp 240-242° C. ¹H-NMR (DMSO-d₆): δ=12.20 (s) 1H, 11.90 (s) 1H, 8.85 (m)2H, 8.60 (d) 1H, 8.40 (d) 1H, 7.70 (m) 8H, 7.30 (m) 5H.

Example 38 Compound 152

Mp 258-259° C. ¹H-NMR (DMSO-d₆), two isomers: 1) δ=12.160 (s) 1H, 11.350(s) 1H, 8.480 (t) 1H, 7.995 (d) 1H, 6.995 (d) 1H, 7.440-7.420 (m) 2H,7.360-7.300 (m) 2H, 7.220-7.020 (m) 5H, 3.805 (s) 3H, 3.695 (s) 3H.

2) δ12.190 (s) 1H) 11.350 (s) 1H, 8.480 (t) 1H, 7.995 (d) 1H, 6.995 (d)1H, 7.440-7.420 (m) 2H, 7.360-7.300 (m) 2H, 7.220-7.020 (m) 5H, 3.762(s) 3H, 3.617 (s) 3H. Example 39 Compound 156

Mp 365-366° C. ¹H-NMR (DMSO-d₆), two isomers: a) δ=13.410 (s) 1H, 11.670(s) 1H, 9.40 (d) 1H, 9.11 (d) 1H, 8.96 (d) 1H, 8.70 (d) 1H, 8.35-8.18(m) 2H, 7.96 (s) 1H, 7.70 (t) 1H, 7.56 (t) 1H, 7.28 (m) 2H.

b) δ=13.290 (s) 1H, 11.67 (s) 1H, 9.305 (d) 1H, 9.095 (d) 1H, 8.960 (d)1H, (d) 1H, 8.70 (d) 1H, 8.495 (d) 1H, 7.87 (d) 1H, 7.70 (t) 1H, 7.56(t) 1H, 7.28 (m) 2H. EI-MS (C₂₃H₁₄N₄O₂)=378.

Example 40 Compound 157

¹H-NMR (DMSO-d₆): δ=13.102 (s) 1H, 11.702 (s) 1H, 8.900-8.840 (m) 3H,8.690 (d) 1H, 8.445-8.400 (m) 3H, 8.301 (d) 1H, 7.747 (t) 1H,7.644-7.624 (m) 1H, 7.622-7.605 (m) 1H, 7.585-7.529 (m) 1H, 7.128-7.086(m) 1H. EI-MS (C₂₃H₁₄N₃F)=351.

Example 41 Compound 160

Mp 320=330° C. ¹H-NMR (DMSO-d₆): δ=13.30 (exc.) 1H, 12.92 (d) 1H, 8.64(d) 1H, 8.56 (d) 1H, 8.43 (m) 2H, 7.62 (m) 4H, 7.34 (t) 1H, 7.03 (d) 1H,5.95 (exc.) 2H.

Example 42 Compound 162

M>400° C. ¹H NM R (DMSO-d₆): δ=13. (s) 1H, 12.25 (s) 1H, 9.41 (d) 1H,9.13 (t) 1H, 8.98 (m) 2H, 8.37 (m) 3H, 7.96 (s) 1H, 7.89 (t) 1H, 7.73(t) 1H, 7.36 (m) 1H. EI-MS (C₂₂H₁₃N₅O₂)=379.

Example 43 Compound 169

Mp 236-237° C. EI-MS: 633.87 (C₃₂H₁₉Br₂N₅) require 633.33. ¹H-NMR(DMSO-d₆), two isomers: 1) δ=13.190 (s) 1H, 11820 (s) 1H, 8.955 (s) 1H,8.910-8.883 (m) 3H, 8.640 (d) 1H, 8.590 (d) 1H, 8.280 (d) 2H, 8.157 (d)1H 7.730

(t) 1H, 8.620 (t) 1H, 7.525 (s) 1H, 7.509 (s) 1H, 7.410 (d) 1H, 7.375(d) 1H. 2) δ=13.190 (s) 1H, 12.060 (s) 1H, 8.955 (s) 1H, 8.910-8.883 (m)3H, 8.640 (d) 1H, 8.590 (d) 1H, 8.280 (d) 2H, 8.157 (d) 1H 7.730 (t) 1H,8.620 (t) 1H, 7.525 (s) 1H, 7.509 (s) 1H, 7.410 (d) 1H, 7.375 (d) 1H.Example 44 Compound 175

EI-MS: 440.11 (C₂₅H₁₈BrN₃ requires 440.07. ¹H-NMR (DMSO-d₆): δ=13.141(s) 1H, 11.779 (s) 1H, 8.842 (d) 1H, 8.673 (s) 1H, 8.636 (s) 1H, 8.508(d) 1H, 8.295 (d) 1H, 8.242 (d) 1H, 7.553 (d) 2H, 7.508 (d) 1H, 7.365(d) 1H, 2.612 (s) 6H.

Example 45 Compound 180

Mp 239-240° C. ¹H-NMR (DMSO-d₆): δ=12.12 (s) 1H, 11.30 (s) 1H, 8.07 (d)1H, 8.03 (d) 1H, 7.93 (d) 1H, 7.72 (d) 1H, 7.65 (m) 2H, 7.54 (t) 1H,7.47 (m) 3H, 7.28 (m) 3H, 7.08 (m) 4H, 2.70 (s) 3H. EI-MS(C₂₈H₂₁N₃)=399.

Example 46 Compound 181

Mp 308-310° C. ¹H-NMR (DMSO-d₆), two isomers: 1) δ=12.547 (s) 1H, 11.570(s) 1H, 8.739 (d) 1H, 8.200-8.790 (m) 3H, 7.695 (m) 2H, 7.570-7.060 (m)10H.

2) δ=12.575 (s) 1H, 11.620 (s) 1H, 8.478 (d) 1H, 8.200-8.790 (m) 3H,7.695 (m) 2H, 7.570-7.060 (m) 10H. ESI-MS (C27H18BrN3)=464. Example 47Compound 182

Mp 264-265° C. ¹H-NMR (DMSO-d₆): δ=12.0 (s) 1H, 11.3 (s) 1H, 8.46 (d)1H, 7.96 (d) 1H, 7.67 (d) 2H, 7.51 (d) 1H, 7.43 (d) 1H, 7.39 (t) 1H,7.28 (t) 2H, 7.15 (m) 1H, 6.62 (d) 2H. EI-MS (C₂₃H₁₈N₄)=350.

Example 48 Compound 183

Mp 240-242° C. ¹H-NMR (DMSO-d₆): δ=12.20 (s) 1H, 11.90 (s) 1H, 8.85 (m)2H, 8.60 (d) 1H, 8.40 (d) 1H, 7.70 (m) 8H, 7.30 (m) 5H.

Example 49 In Vitro Inhibition of Proliferation of Cancer Cells #1

Selected compounds of Formula I were tested for anti-cancer activity invitro using a human colon carcinoma cells (HT-29) and human non-smallcell lung cancer cells (H460). The cells were maintained in α-MEM medium(Wisent, St-Bruno, Qc) supplemented with 10% FBS, and grown at 37° C. inan atmosphere of 5% CO₂. Cells were transferred onto 150 mm tissueculture plates and grown until sub-confluency (70-80%) prior to theiruse.

The anti-cancer activity in vitro was evaluated by a cell proliferationassay based on the ability of live cells to reduce the tetrazolium saltXTT to orange coloured compounds of formazan (XTT cell proliferation kitII, Roche Applied Science, Montreal, QC).

Approximately 4×10³ colon cancer cells (HT-29) or 2×10³ non-small celllung cancer cells (NCI-H460) in 100 μl of complete culture medium wereplated onto 96-well microtiter plates and incubated overnight at 37° C.The medium was then removed by inverting the plate and patting on asterile absorbent cloth. Fifty μl of medium containing the test compoundat either 25 or 100 μM, were added to the wells containing cells andincubated at 37° C. in an atmosphere of 5% CO₂ for 48 h. Followingincubation, 25 μl of an XTT reaction mixture (XTT at a finalconcentration of 0.3 mg/ml) were added to each well and the plates wereincubated for a further 4 h. The absorbance of each sample was thendetermined at a wavelength of 490 nm/650 nm as reference. Each compoundwas tested in duplicate and the results are reported as averages. TableII shows the effect that different compounds of Formula I have on thegrowth of human colon carcinoma HT-29. Table III shows the effect thatdifferent compounds of Formula I have on the growth of human non-smallcell lung cancer cells (H460).

TABLE II Inhibition of Proliferation of Human Colon Carcinoma (HT-29)Cells 100 μM 25 μM Compound % Survival SD(%) % Survival SD(%) 5 110.71.9 110.9 2.8 6 3.2 0.2 11.7 1.6 9 15.1 2.8 68.3 16 10 7.6 0.5 25.8 2.611 94.3 3.6 107.8 1 13 82.4 0.8 105.9 5.4 14 3.8 0.5 55.2 15.7 19 37.17.2 105.5 2.9 20 28.1 5 100 2.7 23 45.7 5.8 98.2 0 25 39.8 4.7 63.9 1.627 35 0.6 62.3 2.4 29 20.9 1 37.1 6.2 31 24.9 1.8 98.6 3.3 32 7.7 0.622.7 0 33 10 0.3 56.1 5.9 34 10.8 0 22.8 2 35 2.5 0.3 44.1 4.4 36 4.70.8 31.6 2.2 38 35.7 1.5 67 7.9 39 53 0.3 96.2 4 40 36.7 1.8 79.1 1.4 421.8 0 59.9 0.1 43 5.7 0.3 28 6.8 44 6.5 0.4 63.6 2.9 45 35 0.6 88.9 3.346 4.5 0 16.1 1 73 62.2 3.2 65 2 83 109.5 4.7 100.3 1.1 CPT-11 51.1 3.282.3 10 Vehicle 100 7 100 7

TABLE III Inhibition of Proliferation of Human Lung Carcinoma (NCI- 460)Cells 100 μM 25 μM Compound % Survival SD(%) % Survival SD(%) 5 106 2.6102 0.7 6 1.9 0.5 10.4 0.8 9 8.4 1.6 98.2 1 10 2.7 0.1 26.9 1.6 11 101.68.3 98.8 3.3 13 96.2 1.1 101.9 4 14 1.8 0.1 83.5 20.4 19 27.3 6.1 89.20.2 20 82.1 20.6 98.6 2.1 23 92.1 0 96.3 0.9 25 89 3.4 99.5 0.8 27 43.11.4 93.5 0.2 29 20.2 1.4 73.8 2.2 31 37.6 5.6 94 2.3 32 2.9 0.5 15 0.233 9.4 2 73.4 2.2 34 7.6 0.9 17.4 0.2 35 1.2 0.1 83.8 8 36 2.4 0.3 24.52.1 38 12.2 1.6 98.8 2 39 17 0.7 98.1 0.3 40 7.7 0.5 97.6 3 42 1.2 0.166.1 16.9 43 3 0.1 18.8 1.5 44 3.4 1 77.3 5.3 45 32.7 5.5 96 1.1 46 1.90.1 11.5 1.4 73 53.5 1.3 89.2 0.6 83 109.2 1.5 100.9 2.6 CPT-11 6.1 0.532.2 4.5 Vehicle 100 3.4 100 3.4

Example 50 In Vitro Inhibition of Proliferation of Cancer Cells #2

The compounds listed below were tested for anti-cancer activity againstseveral carcinoma cell lines as described below and in Examples 51-53.

Cells were maintained in α-MEM medium (Wisent, St-Bruno, QC)supplemented with 10% FBS, and grown at 37° C. in an atmosphere of 5%CO₂. They were transferred onto 150 mm tissue culture plates and grownuntil sub-confluency (70-80%) prior to their use.

The anti-cancer activity in vitro was evaluated by a cell proliferationassay based in the ability of live cells to reduce the tetrazolium saltXTT to orange coloured compounds of formazan (XTT cell proliferation kitII, Roche Applied Science, Montreal, QC). The following cancer celllines were tested: HT-29 colon carcinoma, A498 renal carcinoma, Caki-1renal carcinoma, C8161 melanoma, MDA-MB-231 breast adenocarcinoma, A2058metastatic melanoma, SK-OV-3 ovarian adenocarcinoma, Hep G2 livercarcinoma, AsPC-1 pancreatic adenocarcinoma, PC3 metastatic prostateadenocarcinoma. WI 38 is a human lung fibroblast cell line.

Approximately 2-3×10³ cells in 100 μl of complete culture medium wereplated onto 96-well microtiter plates and incubated overnight at 37° C.,the medium was removed by inverting plate and patting on sterileabsorbent cloth. Fifty μl of medium containing the different compoundsat different concentrations were added and wells were incubated at 37°C. with 5% CO₂ for 48 h. Following incubation, 25 μl of an XTT reactionmixture (XTT at a final concentration of 0.3 mg/ml) were added and wellswere incubated for 4 h. The absorbance of each sample was determined ata wavelength of 490 nm/650 nm as reference. The percentage of survivalwas determined by the ratio between absorbance values of cells incubatedwith the different compounds and their respective controls (cellsincubated with vehicle only). The results are shown in FIGS. 1-4.

FIG. 1 depicts the results with compound 92; FIG. 2 depicts the resultswith compound 28; FIG. 3 depicts the results with compound 50; and FIG.4 depicts the results with compound 42.

Example 51 Concentration Dependence of Inhibition of Cancer CellProliferation by Compound 45 In Vitro

The effect of various concentrations of compound 45 on various cancercell lines was tested following the general protocol outlined in Example50, with the following exceptions. Cell survival was assessed 48 h, 72 hand. 6 days post-treatment by incubating cells with XTT for 2 h. Thecancer cell lines utilised in this example were the same as those listedin Example 50, together with the cervical carcinoma cell line KB. Theresults are shown in FIGS. 5 and 6, which depict cell survival aftertreatment with various concentrations of compound 45. A. 48 h aftertreatment, B. 72 h after treatment and C. 6 days after treatment.

Example 52 Concentration Dependence of Inhibition of Cancer CellProliferation by Compounds 45, 33 and 99 In Vitro

The effect of various concentrations of compounds 45, 33 and 99 on thecolon carcinoma cancer cell line LS513 was tested following the generalprotocol outlined in Example 50, with cell survival being assessed 6days post-treatment. The results are shown in FIG. 7.

Example 53 In Vitro Inhibition of Proliferation of Colon Carcinoma Cells#1

The effect of various compounds of Formula I on the proliferation ofHT-29 colon carconoma cells was tested following the general protocoloutlined in Example 50 with the exception that cell survival wasassessed after 5 to 7 days of treatment. The results usingconcentrations of 2, 10 and 25 μM of each compound are shown in FIG. 8.Results were compiled from different experiments with 5 to 7 days oftreatment The co-efficient of variation for most samples were within 5%.

Example 54 In Vitro Inhibition of Proliferation of Cancer Cells #3

The twenty-three compounds of Formula I shown below were evaluated fortheir antiproliferative effects in a panel of 60 human cancer cell linesas part of the in vitro anticancer screening services provided by theDTP (Developmental Therapeutics Program) of the US National CancerInstitute (NCI) U.S. National Cancer Institute (NCI) of the NationalInstitutes of Health (NIH) in Rockwell, Md. The cancer cell lines usedin this screen are provided in FIG. 9.

The NCI conducts a standard 48/72 hour 60 cell line assay and an invitro time course assay as described in Alley et al. (Cancer Res (1988)48:589). In the standard 60 cell line assay, a minimum of 5concentrations of the test compound are tested at 10-fold dilutionsagainst 60 cell lines and cell growth is assayed at 48 and 72 hoursusing a sulphorhodamine B assay. For the time course analysis, tumourcells are treated with the test compound at various time points, thenwashed and grown in medium free of the test compound until the end ofthe experiment at 144 hrs. This assay employs 20% FBS to betterapproximate the minimum c×t (concentrations and times) test compoundexposure conditions that are required to achieve activity in vivo. Cellgrowth is quantified by an MTT assay (similar to the XTT assay describedabove) and the concentration of the test compound required for growthinhibition is determined. The inhibitory effect of the test compoundsare expressed as a GI₅₀ value, which represents the molar concentrationof the test compound that results in 50% growth inhibition.

All compounds exhibited antiproliferative activity against all humantumour cell lines including NSCLC, leukemia, colon cancer, prostatecancer, melanoma, ovarian cancer, renal cancer, CNS cancer, and breastcancer, with GI₅₀ (growth inhibition by 50%) values ranging from 0.61 μMto 12.3 μM, with an average of 2 μM. The compound 45 had a GI₅₀ value of2.0 μM, while the most effective compound was 90 (FIG. 10A). Thecompounds affected the growth of all cell lines comparatively equally.The average GI₅₀ values for compound 45 ranged from 1.3 μM (renal) to3.4 μM (leukemia) (FIG. 10B). These results suggest that compound 45affects a ubiquitous target. The TGI (total growth inhibition) for thiscompound towards leukemia cell lines was significantly different fromthat of other cell types. These cell lines were not 100% growthinhibited, even at 100 μM, the highest concentration used (FIG. 10C).

Example 55 In Vitro Inhibition of Proliferation of Lung Cancer Cells

The following compounds were tested for their ability to inhibit theproliferation of H460 non-small cell lung carcinoma cells in vitro. Theprotocol described in Example 50 was utilised with the exception thatcell survival was assessed after 6 days of treatment. Each compound wastested at concentrations of 0.2, 2, 10 and 25 μM. The results are shownin FIG. 11.

Example 56 In Vitro Inhibition of Proliferation of Colon Carcinoma Cells#2

The above compounds (as shown in Example 55), together with those shownbelow, were tested for their ability to inhibit the proliferation ofHT-29 colon carcinoma cells in vitro. The protocol described in Example50 was utilised with the exception that cell survival was assessed aftereither 2 or 6 days of treatment. Each compound was tested atconcentrations of 0.2, 2, 10 and 25 μM (compounds 110, 30, 101, 113,103, 107, 108 and 109) or at concentrations of 2.5, 10 and 25 μM(compounds 112, 114, 78, 111 and 45). The results are shown in FIGS. 12and 13. The results shown in FIG. 12 reflect cell survival 6 days aftertreatment with the listed compounds. FIG. 13A shows cell survival 2 daysafter treatment with the listed compounds and FIG. 13B shows cellsurvival 6 days after treatment.

Example 57 Inhibition of Colon Carcinoma Growth In Vivo #1

This Example and the following Example 58 describe in vivo efficacystudies of various compounds of Formula I performed using a mousexenograft model using the human colon adenocarcinoma cell line HT-29.The following compounds were tested.

Groups of five to 10 CD-1 female nude mice (6-7 weeks) were injected inthe lower mid back with human colon adenocarcinoma cells HT-29 (3×10⁶cells in 0.1 ml PBS) subcutaneously, and the treatment initiated 5 dayspost-inoculation (size of tumours=20-40 mm³). The treatment scheduleconsisted of 2×200 μl intraperitoneal injections per day of 5 mg/ml (100mg/Kg/d) for five days and 2 days break, for 4 weeks. Tumour sizes weremeasured during the course of the treatment using calipers, mice werethen sacrificed by cervical dislocation and tumours surgically removedand weighed. FIG. 14 shows the average tumour size (mm³) in thedifferent groups of mice. FIGS. 15 and 16 show the average tumour weightper group of mice and per individual mouse, respectively.

Example 58 Inhibition of Colon Carcinoma Growth In Vivo #2

The protocol described in Example 55 was followed. The results are shownin FIG. 17, which depicts the average tumour size (mm³) in the differentgroups of mice. Abbreviations used in FIG. 17 are as follows:V-ip=Vehicle (i.p); 42 (5-ip)=5 mg/Kg (i.p.); 42 (25-lp)=25 mg/Kg; 42(100-ip); 100 mg/Kg (i.p.); 43 (ip)=100 mg/Kg (i.p.); 45 (ip)=100 mg/Kg(i.p); 44 (ip)=100 mg/Kg (i.p.); 46=100 mg/Kg (i.p); 28=100 mg/kg (i.p);V-op=vehicle, oral; 42 (100-op)=100 mg/Kg (oral).

Example 59 In Vivo Inhibition of Cancer Cell Growth by Compound 45

The ability of compound 45 to inhibit the growth of cancer cells in vivowas further investigated in a mouse xenograft model of hepatocellular(liver) cancer. Groups of five to 10 CD-1 female nude mice were injectedsubcutaneously in the mid right flank with HepG2 human hepatocarcinomacells (1×10⁷ cells). The treatment was initiated 7 days post-inoculationand consisted of 2×200 μl intraperitoneal injections per day (100mg/Kg/d). Tumour sizes were measured during the course of the treatmentusing calipers, and were surgically removed and weighed after 10 weeks.The results obtained are shown in FIG. 18A & B.

Notably, none of the compounds tested in the preceding Examples 57-59showed toxic effects in vivo.

Example 60 Effect of Compound 45 on the Activity of Various Human KinaseEnzymes #1

Compound 45 was tested for its ability to function as a kinase inhibitorusing the kinase profiler service from Upstate Biotechnologies. Thegeneral protocol employed is as follows: recombinant kinases wereincubated with specific substrates, 10 mM MgAcetate, and [γ-³³P-ATP].The reaction was initiated by the addition of MgATP mix. Afterincubation at room temperature for 40 minutes, the reaction was stoppedby the addition of 5 μl of a 3% phosphoric acid solution. 10 μl of thereaction was then spotted on to a P30 filtermat and washed 3 times for 5min. in 75 mM phosphoric acid and once in methanol prior to drying andscintillation counting. Each reaction was performed in duplicate with100 μM ATP −/+10 μM compound 45. Results are presented in Table IV andare expressed as the mean of % control (no compound). PI 3-kinase-γ(PI3K-γ) activity was determined with the PIProfiler™ assay, whichmeasures the binding of the GRPI pleckstrin homology (PH) domain toPIP3, the product of PI3K acting on its physiological substrate PIP2.

Seventy-nine recombinant kinases were tested. Of these 87% retainedgreater then 60% activity in the presence of 10 μM ML-220. Four kinasesretained between 40 and 60% activity (Alk-60%; Aurora-A, 54%; PKD2, 52%;SAPK3, 54%; TrkA, 56%), whereas 5 kinases had less than 40% activity(CaMKII, 32%; PI3Kα, 30%; PI3Kβ, 11%; PI3Kδ, 9%; and PI3Kγ, 22%). Theseresults indicate that compound 45 can function as a kinase inhibitor,and it has a high degree of selectivity for particular kinases.

TABLE IV Kinase Inhibiting Activity of Compound 45 Kinase Family %activity Abl TK 107 ALK TK 60 AMPK CAMK 100 ASK1 STE 99 Aurora-A other54 Ax1 TK 100 BRK TK 112 CaMKII CAMK 32 CaMKIV CAMK 98 CDK1/cyclinB CMGC156 CDK2/cyclinA CMGC 95 CDK2/cyclinE CMGC 117 CDK3/cyclinE CMGC 107CDK6/cyclinD3 CMGC 87 CDK7/cyclinH/MAT1 CMGC 95 CHK1 CAMK 111 CK2 other91 EGFR TK 105 EphA2 TK 95 EphB4 TK 95 ErbB4 TK 73 Fes TK 99 FGFR3 TK 82Fms TK 135 Fyn TK 103 GSK3α CMGC 96 IGF-1R TK 80 IKKβ other 111 IKKαother 150 JNK1α1 CMGC 85 JNK3 CMGC 121 Lyn TK 81 MAPK1 CMGC 85 MAPK2CMGC 99 MAPKAP-K2 CAMK 119 MEK1 STE 88 Met TK 129 MINK STE 91 MKK4 STE96 MKK6 STE 86 MSK1 AGC 76 MST2 STE 77 NEK2 other 90 p70S6K AGC 64 PAK2STE 89 PAR-1Bα CAMK 88 PDGFRα TK 117 PDK1 AGC 106 PI3K□ LIPID 22 PI3K-βLIPID 11 PI3K-α LIPID 30 PI3K-δ LIPID 9 Pim-1 CAMK 70 PKA AGC 83 PKBαAGC 95 PKCμ AGC 90 PKCα AGC 92 PKCδ AGC 87 PKCζ AGC 96 PKD2 CAMK 52 Plk3other 132 PRK2 AGC 83 RAF TKL 100 Ret TK 82 ROCK-II AGC 86 Ros TK 107Rse K 176 Rsk1 AGC 183 SAPK2a CMGC 62 SAPK2b CMGC 80 SAPK3 CMGC 54 SAPK4CMGC 77 SGK AGC 89 SRC TK 102 TAK1 TKL 104 Tie2 TK 109 TrkA TK 56 Yes TK91

Example 61 Effect of Other Compounds of Formula I on the Activity ofVarious Human Kinase Enzymes

To assess whether other compounds of Formula I also affected the samekinases, the inhibitory activity of 10 μM of compound 30 or compound 90was tested on five kinases: Aurora-A, CaMKII, PKD2, SAPK3, TrkA andPI3K. The results are shown in FIG. 19 The results indicated that thesetwo compounds have a different pattern of kinase inhibition thancompound 45.

Example 62 Determination of the Subcellular Localization of Compound 45in Various Cancer Cells

Compound 45 is intrinsically fluorescent, which allowed the subcellularlocalization of this compound to be examined by fluorescent microscopy.Fluorescent microscopy was performed at the Microscopy Imaging Centre,Faculty of Medicine, University of Toronto. Cells were treated with 100μM of compound 45 (FIG. 20A, B, D, E) or 1 μM doxorubicin (FIG. 20C) for1 hour, washed once in PBS, fixed in 3.7% formaldehyde/PBS for 10minutes, washed three times in PBS and mounted with Immuno-fluoro.Images were obtained with a Zeiss laser scanning fluorescent microscopewith an excitation filter range of 360-370 nm (compound 45) or 530-560nm (doxorubicin). For FIGS. 20B and C, differential interferencecontrast images were overlaid with fluorescent images.

Compound 45 localizes to punctuate spots in the perinuclear area ofHT-29 colon adenocarcinoma cells (FIG. 20A), and is excluded from thenucleus and plasma membrane regions (FIG. 20B). In contrast, theanti-cancer agent, doxorubicin, which is also intrinsically fluorescent,is localized in the nucleus (FIG. 20C). A similar localization forcompound 45 was observed in A498 renal carcinoma cells (FIG. 20D) andC8161 melanoma cells (FIG. 20B).

Example 63 Determination of Morphological Changes in Cells Treated withCompound 45

Treatment with compound 45 for 24 hours leads to the formation of largevacuoles within the cytoplasm of HT29 colon adenocarcinoma cells (FIG.21), A498 renal carcinoma cells and MDA-MB-231 breast adenocarcinomacells. These vacuoles are not formed in DMSO- or doxorubicin-treatedcells. Moreover, the nuclear membrane is no longer evident in thephase-contrast images of cells treated with compound 45, even though thenucleus is still intact, as shown by DAPI staining. FIG. 21 showsdifferential interference contrast (DIC) images (top row) andfluorescent images (lower row) of the same cells stained with DAPI, acell permeable marker for the nucleus.

Example 64 Cell Cycle Analysis

The effect of treatment with compound 45 on cell cycle progression inHT-29 colon adenocarcinoma cells was examined by flow cytometry (FIG.22). Values were determined by gate analysis of flow cytometric plotsand are presented in FIG. 22 as a percentage of the total cellpopulation, after eliminating doubles. Apoptotic events inferred by thesurface area preceding G1 phase. Cells were starved for 3 days, andtreated with 15 μM or 25 μM compound 45 for 24 or 48 hours in thepresence of 10% serum, followed by flow cytometric analysis. Treatmentwith compound 45 led to an increase of cells in the G1 phase and adecrease in the S and G2/M phases.

The results presented above in this Example and in Examples 58, 60 and61 indicate that compound 45 suppressed the growth of HT-29 colon cancercells with a GI₅₀ of 2.6 μM, and induced a partial arrest in the G0/G1phase of the cell cycle. Fluorescent microscopy revealed the presence ofcompound 45 within the cytoplasm, but not the nucleus or plasma membraneregions of the cell. In addition, compound 45 was found to inhibitkinase activity in a screen of protein kinases, indicating that thecellular target may be a cytoplasmic protein kinase. These resultsindicate that compound 45 and related derivatives have potential astherapeutic agents for the treatment of human cancer.

Example 65 Selectivity of Compounds of Formula I

Compounds of Formula I that demonstrate the ability to decrease thegrowth or proliferation of at least one cancer cell line may undergofurther testing to evaluate their selectivity towards cancer cells. Anexemplary method to measure the selectivity of the compounds of thepresent invention is provided below.

IC₉₀ values of selected compounds on a panel of normal activelyproliferating cells (HUVEC and WI38) and cancer cells representing colon(HT-29), lung (NCI-H460), breast (MDA-MB-231) and prostate cancer (PC-3)are measured. Compounds with 2-fold or higher overall selectivity to thepanel of cancer cell lines at IC₉₀ are identified as potentialtherapeutics.

IC₉₀ values are determined using the XTT assay as an indicator of growtharrest and/or cytotoxicity. This assay is conducted as outlined inExample 50. Percentage inhibition is calculated for each cell line andIC₉₀ values for each compound and cell type determined. The average IC₉₀values for the normal cells are calculated and divided by the averageIC₉₀ values for the cancer cell lines. Compounds with a selectivityratio of >2 are identified and chosen for further optimization and/ortesting.

Example 66 Additional In Vivo Anti-tumour Efficacy Evaluations

Further pharmacological evaluation of selected compounds is conducted inanimal models of human tumour growth. Data from these studies provideevidence of the therapeutic efficacy of selected compounds againstvarious types of cancer and help to identify compounds with betterpharmacological properties and potency.

Examples of mouse models that can be utilized to investigate theefficacy of selected compounds include, but are not limited to,xenografts of various human tumour types, inoculated subcutaneously intonude mice or mice with severe combined immunodeficiency disorder (SCID)as described above; orthotopic implantation of various human tumours innude or SCID mice for investigation of effects on the tumour in thetarget organ (for example, a pancreatic cancer cell graft implanteddirectly into the pancreas of the animal), and investigation ofspontaneous tumours in normal mice.

In order to provide evidence of the efficacy of a selected compound as asingle agent, it may be evaluated, for example, in specific models(xenograft or orthotopic) for representative human cancers such aspancreas, skin (melanoma), kidney, colon, breast, lung, liver, ovary,prostate, bladder and brain. Similar studies can be conducted toevaluate the performance of test compounds in combination with otherstandard therapeutic modalities used in the treatment of human cancers.

For typical xenograft studies, 5-6 week old, female, CD-1 athymicnude-mice, (Charles River, Montreal, QC) are acclimatized in apathogen-free facility for at least 1 week. Animal protocols followedare in compliance with the Guide for the Care and Use of LaboratoryAnimals in Canada. Approximately 10⁶-10⁷ human tumour cells in 100 mlPBS are subcutaneously injected into the right flank of each mouse. Oncetumours reach an approximate volume of 100 mm³ (several days post tumourcell injection), mice are randomized by tumour size into control andtreatment groups. Test compounds are administered at various doses 5days a week for several weeks. Control animals receive vehicle alone(negative control) and/or a standard chemotherapeutic (positive control)for the same period. The tumour dimensions (length, width, and height)are measured using calipers twice a week over the treatment period.Tumour volume is calculated by the formula L×W×H/2, where L indicateslength, W indicates width and H indicates height. The mice aresacrificed when the tumour burden reaches approximately 10% of totalbody weight and excised tumours are weighed. A standard bar graph isused to demonstrate the differences in tumour weights with each barrepresenting mean tumour weight.

Example 67 Additional Assays to Investigate Potential Mechanism ofAction

The potential mechanism of action of selected compounds can beinvestigated using assays such as cell-cycle analysis, apoptosis assays,anti-angiogenesis assays and immunohistochemical analysis. Arepresentative example of each type of assay is provided below.

i) Cell-cycle Analysis

Alterations in cell cycle are determined using flow cytometric analyses.Tumour cells sensitive to a test compound are synchronized by plating inmedium containing 0.5% FBS for 24 h followed by culturing in FBS-freemedium for 48 h. The cells are then released into complete mediumcontaining 0.1% DMSO (vehicle control) or the test compound at anappropriate concentration (e.g. 3×IC₉₀ value), harvested 16 to 24 hfollowing treatment, washed twice with cold PBS and fixed in 70% ethanolat 4° C. for at least 4 h. The fixed cells are centrifuged at 1500 rpmfor 4 minute at 4° C., washed twice with cold PBS containing 2% FBS,treated with 3 mg/ml ribonuclease (Sigma Chemical Co. Oakville, ON) and50 μg/ml propidium iodide (PI) (Sigma Chemical Co.) for 30 minutes at37° C. The fluorescence of the stained cells is measured using a FACScanflow cytometer and the Cell Quest program (Becton Dickinson, San Jose,Calif.). Data are evaluated using Modfit software (Verity softwareHouse, Topsham, Me.) and the effects of the selected compounds on cellcycle are evaluated.

ii) Apoptosis Assay

DNA fragmentation analysis is used to evaluate the apoptotic effects oftest compounds. Briefly, cells are plated in six-well culture plates 24hr prior to treatment. After incubation with the test compound, mediumcontaining detached cells is transferred to 15 ml conical tubes whilecells still attached to the plate are trypsinized and then added to thesame tubes. After centrifugation, collected cells are washed with PBSand resuspended in 0.5 ml lysis buffer containing 50 mM Tris-HCl, pH8.0, 1.0 M NaCl, 10 mM EDTA and 0.5% SDS. Cell lysates are transferredto microfuge tubes and proteinase K is added to a final concentration of0.2 ml/ml and incubated overnight at 37° C. DNA is extracted byphenol:chlorofonn:isoamyl alcohol (24:24:1), dried and dissolved in 40μM of 10 μM Tris-HCl (pH 8.0) and 0.1 mM EDTA. DNase-free RNase A isadded to each sample for 30 min at 37° C. and 12 μl of each sample areloaded onto a 2% agarose gel containing 0.5 μg/ml ethidium bromide andelectrophoresed. DNA is visualized under UV illumination and theinduction of apoptosis by the test compound is evaluated based on thegeneration of a nucleosomal-size DNA ladder.

iii) Anti-angiogenesis Assay

Proliferation of new capillaries, i.e. angiogenesis orneovascularization, is critical for the transition of a small localizedtumour to expand into a large malignant growth. The Matrigel Plug Assay(see, Passaniti et al. Lab. Invest. (1992) 67:519) is a simple methodfor assessing angiogenesis and the possible anti-angiogenic effect ofselected compounds in mice. Briefly, liquid Matrigel (Becton Dickinson &Co., NJ) is injected subcutaneously near the abdominal midline or thedorsal flank of the animal using a 25-gauge needle. Growthfactor-reduced Matrigel supplemented with 8.3 nM basic fibroblast growthfactor (bFGF, Collaborative Biomedical Products, MA) stays in liquidform at 4° C. bFGF is a proven and potent inducer of angiogenesis. Wheninjected into a mouse (0.5 ml/mouse), Matrigel immediately forms areadily recoverable solid gel, which is removed at various times (notexceeding 10 days) to assess neo-vessel growth around and into the gel.Test compounds are administered according to appropriate doses andschedules. Typically at a 5-day point, mice are sacrificed, overlyingskin is removed and the gels are cut out retaining the peritoneal liningfor support. For quantitation of angiogenesis, two methods areemployed: 1. haemoglobin content in the gel is measured using theDrabkin method (Drabkin and Austin, J. Biol Chem. (1932) 98:719) andDrabkin reagent kit 525 (Sigma, MO); 2. the number of blood vesselsinvading the Matrigel is determined by microscopic analysis after thegels are fixed, embedded in paraffin, sectioned and stained.

iv) Immunohistochemistry

The anti-cancer effects of test compounds can be evaluated in mousexenograft models (as described above) by quantitating the effects ofthese compounds on tumour growth, differentiation, apoptosis andangiogenesis using immunohistochemical methods.

Tumour cell proliferation, angiogenesis and tumour immune infiltratesare delineated immunohistochemically using specific antibodies (Ki-67for proliferation, CD31 for angiogenesis and NK1.1 for NK cells andF4/80 for macrophage). Apoptosis is delineated utilising the TUNEL assay(In Situ Cell Death Detection kit; Boehringer Mannheim, Laval, QC).Signal generation is accomplished by peroxidase catalyzed generation ofenzyme product which is visualized microscopically. Tissue histology isdetermined after H&E staining of separate sections.

Briefly, tumour xenografts from treated mice are isolated, fixed andparaffin embedded individually in blocks and several 5 μm sections arecut for immunostaining and TUNEL assays. One additional section isobtained for H&E. staining. For all immunohistochemical labeling, priorantigen retrieval is employed to improve detection. Typically, a 3-stepamplification method is used to generate signals in immunohistochemistrythat consists essentially of applying a biotinylated secondary antibodythat recognizes the primary antibody, followed by avidin-peroxidaseincubation. The final step is enzyme reaction in stable DAB solution.Immunohistochemical sections are counterstained with hematoxylin fortissue histology. To eliminate non-specific immunostaining with mousemonoclonal antibodies applied to mouse tissues, a specific blocking stepis included in the procedure. Staining patterns are documentedphotographically, examined by at least two independent observers andquantitated by counting a pre-determined number of cells.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification arespecifically incorporated by reference in their entirety to the sameextent as if each such individual patent, publication, and databaseentry were specifically and individually indicated to be incorporated byreference.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1.-17. (canceled)
 18. A method of treating cancer in a mammal,comprising administering to said mammal an effective amount of acompound of formula (VI):

or a pharmacologically acceptable salt thereof, wherein: R4, R5, R7 andR8 are independently hydrogen, halogen, hydroxyl, thiol, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, alkylalkenyl, alkylalkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, substitutedheteroaryl, heteroalkyl, cycloalkyl, substituted cycloalkyl,alkylcycloalkyl, alkylcycloheteroalkyl, nitro, cyano, —CONHHN₂ or—S(O)₀₋₂R wherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl; R6 is hydrogen, halogen, CN, NO₂, HN₂, or —OR, wherein R isC1-C10 alkyl or arylalky; R9 is hydrogen, C1-C10 alkyl, aryl, orhalogen; x is CR11; y is CR12 or N; z is CR13 or N; r is CR14 or N; x′is CR15; y′ is CR16 or N; z′ is CR17 or N; r′ is CR18 or N; R10 is H,lower alkyl, substituted lower alkyl, lower alkenyl, substituted loweralkenyl, lower alkynyl, substituted lower alkynyl, methoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, acyl or —SO₂PhCH₃;R11, R12, R14, R15, R16, and R18 are independently hydrogen, halogen,hydroxyl, thiol, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,alkylalkenyl, alkylalkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, substituted heteroaryl, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, cyano, —N═CRR′, wherein R and R′ areindependently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl,substituted heteroaryl; or —NHC(S)NH-phenyl (substituted orunsubstituted); and, R13 and R17 are independently hydrogen, halogen,hydroxyl, thiol, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,alkylalkenyl, alkylalkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, substituted heteroaryl, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, cyano, —N═CRR′, wherein R and R′ areindependently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl,substituted heteroaryl; or —NHC(S)NH-phenyl (substituted orunsubstituted).
 19. The method of claim 18, wherein said cancer is asolid tumour.
 20. The method of claim 18, wherein said cancer isselected from the group of: breast cancer, central nervous systemcancer, cervical cancer, colon cancer, liver cancer, lung cancer,melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcancer, and leukemia.
 21. The method of claim 18, wherein said compoundis administered in combination with an anti-cancer agent.
 22. The methodof claim 18, wherein said mammal is a human.
 23. The method of claim 18,wherein in the compound of formula VI: R4, R5, R7 and R8 areindependently hydrogen, halogen, hydroxyl, thiol, lower alkyl,substituted lower alkyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido,carboxyl, aryl, substituted aryl, nitro, cyano, —CONHNH₂ or —S(O)₀₋₂Rwherein R is alkyl, substituted alkyl, aryl, substituted aryl,heterocycle, heteroaryl, substituted heterocycle, or substitutedheteroaryl.
 24. The method of claim 18, wherein in the compound offormula VI: x is CR11; y is CR12; z is CR13; r is CR14; x′ is CR15; y′is CR16; z′ is CR17; and r′ is CR18.
 25. The method of claim 18, whereinin the compound of formula VI: x is CR11; y is CR12; z is CR13; r isCR14 or N; x′ is CR15; y′ is CR16; z′ is CR17; and r′ is CR18 or N. 26.The method of claim 18, wherein in the compound is selected from:


27. The method of claim 18, wherein said compound is: